Deposits of low-density proteins in the arterial walls are an important trigger for the development of atherosclerotic disease [15]. Despite increased attention towards this risk factor and the use of pharmacological treatments to lower plasmatic cholesterol levels, atherosclerosis remains one of the principal causes of cardiovascular disease [16, 17]. The apolipoprotein E-deficient (apoE^-/-) mouse, established in 1992, is one of the most important animal models of atherosclerosis [18] because of its propensity to spontaneously develop atheromatous lesions when kept on a cholesterol-rich diet. The apoE is an important modulator of lipoprotein interactions with several receptors, including low-density lipoprotein (LDL) receptors and LDL-related receptor on the surface of several lipoproteins, including chylomicrons, VLDL and HDL particles [19].

ApoE^-/- mice are characterized by increased plasma cholesterol levels and by the development of accelerated atherosclerosis with extensive lipid deposition on major arteries such as the aorta [20]. Similar to what has been observed in human atherosclerotic plaques apoE^-/- mice also have plaques that are heavily infiltrated by CD4⁺ T cells [21]. In these mice atherosclerotic changes of the vessel walls can be observed progressively during aging. Intermediate vascular lesions containing foam cells and smooth muscle cells can be found at 15 weeks, and fibrous plaques appear at 20 weeks of age.

The atherosclerotic process is further accelerated when mice are fed with cholesterol-rich high protein, high carbohydrate and high fat products, the so-called ‘Western diet’ [22]. The histological and morphological analysis of plaque progression and lesion size share similarities with human atherosclerotic lesions [21]. In particular, it has been recently described that old apoE^-/- mice develop plaques in the brachiocephalic arteries that closely resemble human plaques.

Murine studies on atherosclerotic lesions are nonetheless difficult as cholesterol metabolism, lipid profile, cardiovascular physiology, plaque pathology are different to that of humans. Although apoE^-/- mice display vascular atherosclerotic changes they do not develop spontaneous thrombotic occlusions and/or embolic events [22]. Despite these limitations of the apoE^-/- mouse, it has been possible to study cholesterol absorption inhibitors such as ezetimibe [23], antihypertensive drugs, such as angiotensinogen converting enzyme inhibitors or angiotensin II receptor blockers [24], and antidiabetic drugs as rosiglitazone and trogliatiazone [22, 25]. This demonstrates once again that quite different risk factors contribute to plaque formation and that these can be addressed therapeutically.

Another important animal model in the study of hypercholesterolemia is the Apolipoprotein B (apoB) mouse. ApoB is the major protein directing the metabolic fate of both VLDL and LDL, and its increased plasma concentrations are associated with the development of cardiovascular diseases. The transgenic apoB mice that express human apoB have similar plasma concentrations to those of normolipidemic humans when fed on normal diet [26]. However when these mice are fed with the typical 'Western Diet' pronounced atherosclerosis develops with a higher extent in C57BL/6 female mice than males. Macrophage foam cells characterize primarily the atherosclerotic lesions that form [27].

While in humans the deficiency of LDL receptors (LDLR) leads to a dramatic phenotype of pronounced hypercholesterolemia with catastrophic clinical consequences, mice lacking the LDL receptor (LDLR^-/-) display only a modest hypercholesterolemia when fed on a normal diet [28]. As in the other model of hypercholesterolemia (apoE or apoB mice) also these mice develop atherosclerosis on the Western type diet. The lesions formed in these mice are of simple morphology and predominantly consist of lipid-laden macrophages [29].

A key component to the formation of the atherosclerotic lesion, which involves an inflammatory process, has also been studied extensively and targeted with various anti- inflammatory drugs (acetylsalicylic acid, sulindac, celecoxib) [30-32]. Mixed results on plaque size and progression have been observed in these studies and up to now it is not entirely clear whether anti-inflammatory drugs may have a beneficial role on the atherosclerotic plaque in humans [22].

Other experimental studies on atherosclerosis have used the apoE^-/- mouse as starting basis for double knockout mice or transgene to study genes involved in lipid metabolism (e.g. acyl CoA cholesterol acyl transferase-2 [33], ATP binding cassette (ABC) transporter ABCA1 [34], low density lipoprotein receptor [35]), inflammation (e.g. C-reactive protein [36], inducible nitric oxide synthase (iNOS) [37], endothelial nitric oxide synthase (eNOS)[38], interleukin-1 beta [39]) and blood hemostasis (e.g. fibrinogen [40], plasminogen [41], plasminogen activator inhibitor-1 [42]). See also reference [43] for an extensive review on atherosclerosis mouse models.

Arterial hypertension, defined as untreated systolic pressure of ≥140 mmHg, diastolic pressure of ≥90 mmHg or prescription of antihypertensive drugs, affects nearly one in three adults in the United States. Blood pressure, particularly systolic blood pressure, increases with age [44]. Hypertension can be subdivided into two main categories: primary hypertension and secondary hypertension. Primary, essential or idiopathic hypertension is assumed when no specific cause can be found. In secondary hypertension, elevated blood pressure levels are due to specific reasons (most often renal dysfunction, hormone alterations and/or sleep apnea) that if identified can be corrected [45].

Hypertension is the most important risk factor for stroke and a direct correlation between blood pressure values and stroke risk has been described [46, 47]. Subjects with blood pressure lower than 120/80 mmHg have about half the lifetime risk of stroke compared to subjects with higher blood pressure [48]. The relationship between blood pressure and cardiovascular risk is considered to be continuous, consistent and independent of other risk factors. [49].

Although recent advances in blood pressure control (data from the National Health and Nutrition Examination Survey, NHANES 1999–2004) showed that of the subjects with hypertension aged 18 and older, 71.8% were aware of their condition, only 61.4% were under current treatment and 35.1% had arterial pressure levels that were reasonably controlled (data from National Center for Health Statistics and National Heart, Lung and Blood Institute). Because of the high prevalence of hypertension and its high direct and indirect costs (estimated to be of $69.4 billion for 2008) identifying genetic and environmental risk factors is not only important for the prevention of CVD but also for the development of new pharmacological tools aimed at preventing late complications [50].

Over the past 50 years various animal models of hypertension have been developed, predominantly in the rat. Different rat models can either be grouped according to their capacity to mimic the etiology of hypertension (primary vs. secondary and genetic vs. environmental) or according to the resulting end-organ damage, cardiac failure vs. renal failure vs. vascular injury. With the use of these models, analysis of genetic susceptibility factors to multifactor diseases has become feasible as animal studies avoid (1) genetic heterogeneity, (2) environmental noise, and (3) the poor statistical power related to small patient populations [51].

Spontaneously hypertensive rats (SHR) represent the animal model most widely used in hypertension studies. They mimic in particular the human form of primary hypertension although the Mendelian type of inheritance that characterizes hypertension in the SHR is rarely encountered in humans. Okakamoto and Aoki [52] obtained the SHR rats by inbreeding Wistar rats showing the highest blood pressure. These animals develop increases in blood pressure beginning at six to seven weeks of age and reach a stable level of hypertension (reach systolic blood pressures of 180- 200 mmHg) by 17 to 19 weeks.

The SHR develop many features of hypertensive end-organ damage as cardiac hypertrophy, present in 30% of cases [53], heart failure present in 60% of the rats at the age of 18- 24 months, renal proteinuria, decreased creatinine clearance as signs of renal dysfunction [54, 55]. However, they do not exhibit gross vascular problems, apart from consistently impaired endothelium relaxation [56]. They have no tendency to develop strokes, macroscopic atherosclerosis or vascular thrombosis. Mean survival of this strain is of 10- 21 months. Interestingly various anti-hypertensive drugs like calcium antagonists, inhibitors of the renin- angiotensin system and direct vasodilators can lower consistently blood pressure in the SHR strain. However end organ damage seems not to be prevented by any kind of treatment [57].

Another strain, the spontaneous hypertensive stroke prone rat(SHR-SP) exhibits an early onset of hypertension, and 80% of animals experience stroke between 9 and 13 months of age [58]. There are many pathogenetic similarities between strokes in SHR-SP rats and humans [59]. On the SHR-SP, it was in fact possible to identify one of the genetic components responsible for large infarct volumes in response to a focal ischemic insult, independently from blood pressure values. The genes encoding atrial and brain natriuretic factor were identified in this case. These genes have well- established vasoactive properties and might be involved in the control of vasoreactivity, growth and remodeling of cerebral vessels [60].

Further, the SHR-SP exhibits salt-sensitivity, increased vascular release of super oxide and decreased total plasma antioxidant capacity. The super oxide release in the SHR-SP rats has been found to inactivate nitric oxide in mesenteric arteries and to be directly correlated with hypertension. Interestingly, administration of super oxide dismutase (SOD) returns the bioactive nitric oxide levels to normal together with blood pressure [61]. These data suggest that oxidative stress plays an important role in severe hypertension in salt-loaded SHR-SP rats and its associated vascular damage [62].

To help to clarify the role of individual genes, other animal models of hypertension were developed. The transgenic rat overexpressing the mouse Ren-2 gene ((TGR-mREN2)27) is important in hypertension research as, in contrast to the SHR and SHR-SP rat, it is a genetically inherited form of hypertension for which the responsible gene is known. Nonetheless the exact mechanism via which this mutation leads to hypertension is not clear. That hypertension usually depends not on a single candidate gene but on a variety of genes can be clearly shown by the fact that the severity of hypertension in (TGR-mREN2)27 rats is dependent on the genetic background. For example, Sprague- Dawley rats develop an accelerated and more malignant hypertension compared with Lewis rats that do not develop hypertension [63].

A further animal model of primary genetic hypertension is the Dahl salt- sensitive rat. This rat develops severe and fatal hypertension when it receives a high salt diet. The increased sensitivity to salt is also present when these rats are fed with normal diet as they also develop hypertension. Genetic analysis has revealed a role for the angiotensin converting enzyme (ACE) and atrial natriuretic factor (ANP) receptor genes. Cardiac hypertrophy develops in about 32% of rats and cardiac heart failure evolves early after its onset (about 4-5 months of age) [64, 65].

To model secondary renal hypertension in the rat the most used method is to occlude the renal artery with a clip, leaving either the controlateral kidney intact (‘two-kidneys one-clip’) or removing this second kidney (‘one-kidney one-clip) [66, 67]. Although clipping of the renal artery has also been used in the dog, the rat model is much more reliable. In the rat model, like in human disease, hypertension is chronic, while in the dog the rapid formation of vessel collaterals accounts for its short duration. Cardiac hypertrophy that affects approximately 25-50% of rats subjected to the clipping can be attenuated by administration of renin- angiotensin inhibitors and calcium antagonists, but diuretics and beta-blockers are not effective. As such, animal models of hypertension offer the opportunity to investigate not only the pathophysiology of hypertension, but also to the effects of anti-hypertensive drugs. Further, they can be used for inducing stroke, as has been made with the SHR-SP rat, to study the complex vascular response that follows stroke [60, 68].

High levels of plasma homocysteine represent a relevant risk factor for cardiovascular disease, stroke, and venous thromboembolism [69, 70]. Although severe forms of hyperhomocysteinemia (plasma concentrations above 100 µmol L^-1) may result from mutations of enzymes involved in the metabolism of homocysteine such as cystathionine β-synthetase (CBS) or 5,10-methylenetetrahydrofolate reductase (MTHFR), also the deficiency of cofactors (folic acid, vitamin B6 and B12) can result in the mild forms of hyperhomocysteinemia characterized by a plasma concentration of 12–50 µmol L^-1. Patients affected by severe forms of hyperhomocysteinemia have, if untreated, 50% of chance of developing a major vascular event (myocardial infarction, stroke, or venous thromboembolism) before the age of 30 years [71].

Also mild hyperhomocysteinemia, as pointed out by recent epidemiological studies, is considered as a risk factor for stroke, cardiovascular disease, and venous thromboembolism [72].

The underlying pathophysiological mechanism of hyperhomocysteinemia has been suggested to be enhanced atherothrombosis. Homocysteine causes in fact endothelial cell dysfunction and induces apoptosis in endothelial and smooth muscle cells. The strongest evidence that homocysteine plays a causative role in atherothrombosis has been gained from studies of hyperhomocysteinemia in animal models in the last ten years.

The first genetic model of hyperhomocysteinemia in mice was developed in 1995 through targeted disruption of the CBS gene thereby establishing a severe form of hyperhomocysteinemia induced by homozygous CBS-deficiency (plasma levels of homocysteine elevated by 40-fold with plasma concentrations above 200 µmol L^-1) and a mild form in heterozygous CBS deficiency (plasma concentration 6-15 µmol L^-1) [73]. Severe growth retardation, hepatic steatosis and dislocation of the lens are characteristics of the homozygous mouse model. Very similar clinical features characterize also the natural history of patients with homocystinuria [74]. On the other hand, heterozygous mice with twice the normal plasmatic homocysteine levels are apparently healthy, thereby making them interesting models for studying mild forms of hyperhomocysteinemia. Notably heterozygous CBS- deficient mice display endothelial dysfunctions but do not show increased risk for atherosclerotic plaque formation [73].

Another model of hyperhomocysteinemia is the MTHFR-deficient mouse. In contrast to the CBS-deficient mouse, heterozygous and homozygous MTHGR-deficient animals show early atherosclerotic lesions. Despite the presence of atherosclerotic plaques, these mice do not exhibit increased lipid plasma levels, thereby suggesting that hyperhomocysteinemia contributes indirectly to the development of atherosclerosis [75].

In atherosclerosis prone animals like apoE^-/- mice, hyperhomocysteinemia accelerates the development of atherosclerosis through the enhanced expression of inflammatory mediators (e.g. receptor for advanced glycation end products (RAGE), vascular cell adhesion molecule-1 (VCAM-1), tissue factor (TF), and matrix metalloproteinase-9 (MMP-9) [76]) and by the dysregulation of lipid metabolism [77]. A major goal of current research using these animal models is to uncover the molecular and cellular mechanisms underlying the atherogenic effects of hyperhomocysteinemia and to develop efficacious treatments for lowering the increased risk of hyperhomocysteinemic patients [78, 79]. In fact, although today the homocysteine level can be reduced through adequate diet, no proof is available that lowering mild elevated homocysteine levels as primary prevention decreases the cerebro- or cardiovascular risk. (The Heart Outcomes Prevention Evaluation, HOPE 2 Investigators 2006) [80]. Also in the secondary prevention for stroke there are currently no recommendations to treat elevated homocysteine levels with vitamins or folic acid [81, 82]. We hope that the large Australian study (VITATOPS) in the next few years with their data together with increased insight in the pathogenesis of hyperhomocysteinemia will make the image round.