**3. Cholesterol—factor of endothelial senescence and endothelial dysfunction**

Senescence of endothelial cells is known to mediate the endothelial damage that occurs during the initial phase of atherosclerosis. Aging cells of the vascular wall lead to endothelial dysfunction, resulting in the synthesis of inflammatory cytokines and promoting the progression of atherosclerosis. The second stage of developing atherosclerosis, fibrous plaque formation, is characterized by increased lipid accumulation in the intima, resulting in fibrous tissue proliferation and vitreous degeneration, forming characteristic plaques in the intima. Also, macrophages accumulate in the subendothelial space, where they induce pathology by increasing the expression of key atherogenic and inflammatory cytokines and chemokines [14]. In the third stage, atherosclerotic plaque formation, the fibrous tissue is large and necrotic, enriched in lipids, while the lesion surface is thinner and few foam cells are present at the base and margin. In atherosclerotic lesions, smooth muscle cells of the vascular wall migrate from the media to the intima, accumulate around the lipid core formed by dead foam cells and switch from a contractile to a synthetic phenotype. Macrophages, on the other hand, which phagocytized lipids, display an abnormal or activated phenotype, which promotes pathological vascular proliferation [15]. At this stage, proliferation dominates the smooth muscle cells of the vascular wall, but aging does not occur and a typical atherosclerotic plaque is formed. The fourth stage involves

changes secondary to atherosclerotic plaques, in which aging macrophages promote plaque instability, degradation of elastic fibers and thinning of the fibrous cap, as well as increased expression of metalloproteases and formation of ulcers and thrombi [14]. At this stage, foam cells induce senescence of human vascular endothelial cells by releasing 4-hydroxynonenal (4-HNE) [16], which exacerbates senescence and induces atherosclerosis. Senescent human vascular wall smooth muscle cells differentiate into an osteogenic phenotype and undergo expression of calcifying factors, which eventually leads to calcification of the atherosclerotic plaque. It is noteworthy that human vascular smooth muscle cells proliferate in the early phase of atherosclerotic plaque formation. However, the proliferation rate of these cells is lower in advanced plaques than in early lesions, indicating that cell senescence may occur [17]. In addition, vascular injury and phenotypic transformation of senescent human vascular wall smooth muscle cells also play a role in mediating vascular calcification [18]. Cellular aging is not a consequence of a single cause, but there are many factors that can induce cellular aging. Premature cellular aging, can be caused by factors such as miRNAs, homocysteine, hyperglycaemia, hypertension, hyperlipidaemia, hyperphosphataemia and oxidative stress, by reducing telomerase activity, increasing ROS production and promoting vascular calcification, mitochondrial dysfunction and DNA damage.

High cholesterol and triglyceride levels have also been found to be associated with an increased risk of atherosclerosis and shorter life expectancy. In fact, the vascular endothelial dysfunction that occurs during human aging is the factor, and the accumulation of lipids in the vascular endothelium activates leukocytes to produce cytokines and chemokines that recruit macrophages. On the other hand, macrophages enhance the inflammatory response and secrete vascular endothelial growth factor, a key cytokine that mediates angiogenesis and the inflammatory response. And hyperlipidaemia itself is a major risk factor for aging, hypertension and diabetes.

The relationship between hypercholesterolemia, atherosclerosis and aging is still poorly understood. Low-density lipoprotein (LDL) (cholesterol) in general is an important physiological compound for cellular function, but in high concentrations can lead to atherosclerosis. It is generally accepted that the oxidized form of cholesterol leads to endothelial dysfunction, which is the initial step in the formation of atherosclerotic plaques. Oxidized low-density lipoprotein acts by binding to multiple scavenger receptors (SRs), such as SR-AI, SR-A2, and can also increase the expression of endothelial cells' own LOX-Ion receptor and activate these cells [19–21]. Under physiological conditions, endothelial cells secrete many factors, monitor the transport of plasma molecules and regulate vascular tone. In addition, endothelial cells are involved in the regulation of cholesterol and lipid homeostasis, signal transduction, immunity and inflammation [22]. And, in addition, oxidized low-density lipoprotein promotes the growth and migration of smooth muscle cells, fibroblasts and macrophages. Vascular lesions are most often caused by hypercholesterolaemia, which can be induced by dietary supplementation, overproduction of lipoproteins by the liver or genetic mutations of lipid receptors and other proteins that regulate lipid homeostatic pathways.

### **3.1 Mutations of genes regulating the cholesterol level**

Hypercholesterolaemia is a common, and still underdiagnosed, autosomal dominantly inherited disorder that is estimated to occur at a prevalence of ≈1 in 220 people worldwide. Familial hypercholesterolaemia (FH) is characterized by a persistent lifelong elevation of low-density lipoprotein cholesterol (LDL-C) and, if untreated, leads to early onset atherosclerosis and an increased risk of cardiovascular

#### *Genetic Markers of Endothelial Dysfunction DOI: http://dx.doi.org/10.5772/intechopen.109272*

events. Untreated hypercholesterolaemia in men and women is associated with a very high risk ranging from 30–50% of having a fatal or non-fatal cardiac event at 50 and 60 years of age, respectively [23]. The most common cause of single-gene familial hypercholesterolaemia is pathogenic variants in the LDL receptor gene (LDL-R), which account for 85–90% of genetically confirmed cases of familial FH. Pathogenic variants in the gene for apolipoprotein (ApoB), a ligand for the LDL receptor, a component of LDL resulting in reduced binding of LDL to LDL-R, or gain-of-function mutations in the gene for proprotein convertase subtilisin/kexin 9 (PCSK9), resulting in increased destruction of LDL-R, account for 5–15% and 1% of cases of monogenic hypercholesterolaemia, respectively [24]. There is also an autosomal recessive form of hypercholesterolaemia in the human population, caused by homozygous mutations in the LDL-R adaptor protein which, is associated with the mild phenotype of homozygous hypercholesterolaemia found in Sardinian residents [25].

With the exception of the homozygous form of familial hypercholesterolemia (HoFH), FH is generally a silent disease. HoFH usually manifests with pathognomonic physical symptoms in childhood, such as cantelosis, tendon xanthoma and corneal arching. FH is diagnosed clinically based on a weighted combination of physical findings, personal or family history of hypercholesterolemia, early ischemic disease in the family and circulating LDL-C levels. The genetic cause is highly heterogeneous. Mutations in the LDL receptor genome are very common and occur at different sites disrupting receptor function in different ways. They therefore have different pathological significance. The spectrum of functional alterations in APOB outside the fragments routinely screened is growing. The ClinVar database at NCBI shows all the mutations in this gene described to date. There are about 3000 of them, and of these mutations that are labeled as pathological there are about 1000. They are mainly missense, nonsense frameshift mutations including about 500 deletions and 170 duplications. The largest number of known mutations are single nucleotide mutations mainly in coding regions of the gene, about 2000.

The known spectrum of mutations in APOB has been increasing in recent years thanks to next-generation sequencing (NGS) techniques, which allow all 29 exons of APOB to be studied without increasing laboratory workload [26–29]. However, as APOB is a highly polymorphic gene, these variants require functional assessment before a clear diagnosis can be made [27]. It is also known that mutations in the APOB gene do not have 100 per cent penetrance, and the phenotype of patients is usually milder than in patients with FH caused by LDLR mutations [30].

The ClinVar database from NCBI is being updated with known pathological mutations in the APOB gene. There are currently 84 of them, most of which are located in the hydrophilic part of the apoB protein, the part that can bind to the LDL receptor. Mutations of the nonsense, missense and reading frame shift types dominate among the pathologies leading to familial hypercholesterolemia.

Familial hypercholesterolemia (FH), a major risk factor for coronary artery disease (CAD), is typically caused by mutations in genes that code for proteins responsible for removing low density lipoprotein (LDL) from the circulation. Only 17 pathogenic mutations in the PCSK9 gene are currently known and presented in the ClinVar database from NCBI. PCSK9 was discovered in 2003 when gain-of-function (GOF) mutations in this gene were identified as causative of FH in an autosomal dominant manner [31]. These GOF mutations are associated with hypercholesterolemia and a higher risk of CAD [32–36]. For example, a mutation in the apoB gene p.S127R is specifically associated with overproduction of this protein, resulting in greater synthesis of very low-density lipoprotein (VLDL), intermediate-density lipoprotein

(IDL) and, consequently, LDL [32]. Another mutation of this gene p.E670G is associated more with serum lipid parameters, including total cholesterol (TC), high-density lipoprotein (HDL) and Apo B [33], as well as with an increased risk of stroke due to large vessel atherosclerosis and ischaemic stroke [36]. Serum PCSK9 levels, has been identified as a major predictor of carotid atherosclerosis independent of other risk factors in asymptomatic patients [37]. Furthermore, the contribution of PCSK9 concentrations to FH severity appears to be independent of LDL receptor genotype [38]. Recently, a homozygous gain-of-function mutation of the PCSK9 gene was characterized that is associated with the phenotype of a patient whose cholesterol is 316 mg/ dl and LDL was 234 mg/dl at the age of 11 years [39]. This patient has no mutations in the LDL receptor or Apo B genes [39].

Loss-of-function mutations of the PCSK9 gene are associated with hypocholesterolaemia and significant protection against CAD [40–43]. Notably, the p.Y142X mutation is found only in 0.4 per cent of African Americans, but not in other ethnic groups [40]. The p.C679X mutation is more common in African Americans and Zimbabwean Africans, but very rare in European Americans [41]. One individual has been described who is homozygous for the p.R46L mutation and has a total cholesterol level of 11 mg/ dl [42]. In one family, six of the eight members who carry the p.R46L mutation have LDL levels below the bottom 10% percentile of LDL [42]. Another study reported that two healthy women with 'loss of function' mutations affecting both alleles of the PCSK9 gene have extremely low LDL cholesterol levels (14 mg/dL) [41–43].

The concept of polygenic hypercholesterolaemia for patients with a clinical diagnosis of FH but no monogenic cause was presented in 2013 by Talmud et al. [44]. This concept is based on the cumulative effect of LDL-C-raising alleles with a cumulative effect, perhaps in a complex interaction with the environment that leads to an increase in LDL-C, producing an FH-like phenotype and presenting this type of hypercholesterolaemia as a typical complex disease.

The more often publishing genes with polymorphisms contributing to the high cholesterol phenotype include cadherin EGF LAG 7-pass G-type receptor 2, ATP-binding cassette subfamily G members 5 & 8 (ABCG5/8), sterol regulatory element binding protein-2 (SREBP-2), signal transducing adaptor family member 1 (STAP1), and Apo E. Talmud's group developed a genetic risk score (GRS) based on scoring 12 SNPs where individuals above the top decile of the distribution of LDL-C scores were described as having a higher probability of polygenic hypercholesterolaemia [44]. Then, by removing SNPs with smaller effects/lower frequencies, they showed that a weighted score of six SNPs performed as well as a score of 12-SNPs. The top three quartiles of the distribution also indicated a greater likelihood of a polygenic explanation for their elevated LDL-C [45]. Another study established the 10-SNP GRS, which showed a strong association with high LDL cholesterol, confirming the validity of this score as a genetic risk marker for elevated LDL cholesterol [46]. In this cohort, individuals with an extreme weighted GRS ≥1.96 (≥90th percentile) were defined as having polygenic severe hypercholesterolaemia. Research has gone further and a study of patients with severe hypercholesterolaemia found that a high polygenic score for 2 million-SNP LDL-C (upper 5th percentile) could explain hypercholesterolaemia in up to 23% of patients, while only 2% carried a monogenic mutation [47].

With the development of genetic testing in recent years, a mutation in any of the three known autosomal dominant genes causing familial hypercholesterolaemia is found in the majority of cases with a clinical diagnosis of familial hypercholesterolaemia. SituationBecause individuals with polygenic background hypercholesterolaemia do not have the same inheritance pattern observed in monogenic familial

### *Genetic Markers of Endothelial Dysfunction DOI: http://dx.doi.org/10.5772/intechopen.109272*

hypercholesterolaemia, familial cascade screening is not recommended for individuals with polygenic background, as only 30% of relatives have elevated LDL-C levels compared to 50% in monogenic families. The presence of a causative monogenic mutation is associated with the highest cardiovascular risk vs. no mutation or polygenic ancestry, providing prognostic information independent of LDL-C. This may also help to assess the intensity of intervention. Treatment adherence also appears to be higher after monogenic confirmation of hypercholesterolaemia.
