Premature atherosclerosis: An analysis over 39 years in the Netherlands. Implications for young individuals in high-risk families.

Cardiovascular disease (CVD), especially atherosclerotic cardiovascular disease (ASCVD), is one of the most important disease problems in the world accounting for an estimated 18.6 million deaths globally. Although older individuals are more often affected, ASCVD event at a young age is of particular importance because of more healthy years lost. Therefore, it is important to identify young individuals correctly at risk of ASCVD events in an early stage. Unfortunately, current risk score algorithms underestimate ASCVD event risk at a younger age. Both coronary artery calcium scoring (CACs) and family history of premature ASCVD (FH-PASCVD) have emerged as reliable screening tools to be able to identify individuals at risk for ASCVD events. Positive FH-PASCVD is associated with higher absolute CAC scores in first-degree 'healthy' family members and the proportion of individuals above the CACs percentile threshold to warrant treatment is also higher as compared to the general population. Therefore, a positive FH-PASCVD identifies so-called high-risk families and adding CAC scoring within these families identifies individuals at increased risk for ASCVD events. In individuals from high-risk families with an elevated CAC score, ASCVD events can be prevented when treated with statins and aspirin. Therefore, we suggest assessing FH-PASCVD in young 'healthy' individuals as a first screening step and subsequently performing CAC scoring to be able to start treatment at an early stage, since not only the lower is better, but also the earlier is better.

Netrin-4 expression by human endothelial cells inhibits endothelial inflammation and senescence.

Netrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear. We found that the expression of netrin-4 is highly regulated in endothelial cells and is important for quiescent healthy endothelium. Netrin-4 expression is upregulated in endothelial cells cultured under laminar flow conditions, while endothelial cells stimulated with tumor necrosis factor alpha resulted in decreased netrin-4 expression. Targeted reduction of netrin-4 in endothelial cells resulted in increased expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Besides, these endothelial cells were more prone to monocyte adhesion and showed impaired barrier function, measured with electric cell-substrate impedance sensing, as well as in an 'organ-on-a-chip' microfluidic system. Importantly, endothelial cells with reduced levels of netrin-4 showed increased expression of the senescence-associated markers cyclin-dependent kinase inhibitor-1 and -2A, an increased cell size and decreased ability to proliferate. Consistent with the gene expression profile, netrin-4 reduction was accompanied with more senescent associated ß-galactosidase activity, which could be rescued by adding netrin-4 protein. Finally, using human decellularized kidney extracellular matrix scaffolds, we found that pre-treatment of the scaffolds with netrin-4 increased numbers of endothelial cells adhering to the matrix, showing a pro-survival effect of netrin-4. Taken together, netrin-4 acts as an anti-senescence and anti-inflammation factor in endothelial cell function and our results provide insights as to maintain endothelial homeostasis and supporting vascular health.

Genetic variants in SUSD2 are associated with the risk of ischemic heart disease.

BACKGROUND: Genetic factors partly determine the risk for premature myocardial infarction (MI). OBJECTIVES: We report the identification of a novel rare genetic variant in a kindred with an autosomal dominant trait for premature MI and atherosclerosis and explored the association of a common nonsynonymous variant in the same gene with the risk of ischemic heart disease (IHD) in a population-based study. METHODS: Next-generation sequencing was performed in a small pedigree with premature MI or subclinical atherosclerosis. A common variant, rs8141797 A>G (p.Asn466Ser), in sushi domain-containing protein 2 (SUSD2) was studied in the prospective Copenhagen General Population Studies (N = 105,408) for association with IHD. RESULTS: A novel heterozygous nonsense mutation in SUSD2 (c.G583T; p.Glu195Ter) was associated with the disease phenotype in the pedigree. SUSD2 protein was expressed in aortic specimens in the subendothelial cell layer and around the vasa vasorum. Furthermore, the minor G-allele of rs8141797 was associated with per allele higher levels of SUSD2 mRNA expression in the heart and vasculature. In the Copenhagen General Population Study, hazard ratios for IHD were 0.92 (95% CI: 0.87-0.97) in AG heterozygotes and 0.86 (0.62-1.19) in GG homozygotes vs noncarrriers (P-trend = .002). Finally, in meta-analysis including 73,983 IHD cases and 215,730 controls, the odds ratio for IHD per G-allele vs A-allele was 0.93 (0.90-0.96) (P = 4.6 × 10-7). CONCLUSIONS: The identification of a truncating mutation in SUSD2, which was associated with premature MI and subclinical atherosclerosis, combined with the finding that a common missense variant in SUSD2 was strongly associated with a lower risk of IHD, suggest that SUSD2 may alter the risk of atherosclerosis.

The identification and function of a Netrin-1 mutation in a pedigree with premature atherosclerosis.

BACKGROUND AND AIMS: Neuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we investigated the role of a c.1769G > T variant in Netrin-1 in (premature) atherosclerosis. METHODS: To determine the effect of the genetic variation, purified Netrin-1, either wild type (wtNetrin-1) or the patient observed variation (mutNetrin-1), was used for migration, adhesion, endothelial barrier function and bindings assays. Expression of adhesion molecules and transcription proteins was analyzed by RT-PCR, Western blot or ELISA. To further delineate how mutNetrin-1 mediates its effect on cell migration, lenti-viral knockdown of UNC5B or DCC was used. RESULTS: Bindings assays revealed a decreased binding capacity of mutNetrin-1 to the receptors UNC5B, DCC and ß3-integrin and an increased binding capacity to neogenin, heparin and heparan sulfate compared to wtNetrin-1. Exposure of endothelial cells to mutNetrin-1 resulted in enhanced monocyte adhesion and expression of IL-6, CCL2 and ICAM-1 compared to wtNetrin-1. In addition, mutNetrin-1 lacks the inhibitory effect on the NF-κB pathway that is observed for wtNetrin-1. Moreover, the presence of mutNetrin-1 diminished migration of macrophages and smooth muscle cells. Importantly, UNC5B or DCC specific knockdown showed that mutNetrin-1 is unable to act through DCC resulting in enhanced inhibition of migration. CONCLUSIONS: Our data demonstrates that mutNetrin-1 fails to exert anti-inflammatory effects on endothelial cells and more strongly blocks macrophage migration compared to wtNetrin-1, suggesting that the carriers of this genetic molecular variant may well be at risk for premature atherosclerosis.

Netrin-1 and the Grade of Atherosclerosis Are Inversely Correlated in Humans.

OBJECTIVE: Netrin-1 has been shown to play a role in the initiation of atherosclerosis in mice models. However, little is known about the role of Netrin-1 in humans. We set out to study whether Netrin-1 is associated with different stages of atherosclerosis. Approach and Results: Plasma Netrin-1 levels were measured in different patient cohorts: (1) 22 patients with high cardiovascular risk who underwent arterial wall inflammation assessment using positron-emission tomography / computed tomography, (2) 168 patients with a positive family history of premature atherosclerosis in whom coronary artery calcium scores were obtained, and (3) 104 patients with chest pain who underwent coronary computed tomography angiography imaging to evaluate plaque vulnerability and burden. Netrin-1 plasma levels were negatively correlated with arterial wall inflammation (ß, -0.01 [95% CI, 0.02 to -0.01] R2, 0.61; P<0.0001), and concentrations of Netrin-1 were significantly lower when atherosclerosis was present compared with individuals without atherosclerosis (28.01 versus 10.51 ng/mL, P<0.001). There was no difference in Netrin-1 plasma concentrations between patients with stable versus unstable plaques (11.17 versus 11.74 ng/mL, P=0.511). However, Netrin-1 plasma levels were negatively correlated to total plaque volume (ß, -0.09 [95% CI, -0.11 to -0.08] R2, 0.57, P<0.0001), calcified plaque volumes (ß, -0.10 [95% CI, -0.12 to -0.08] R2, 0.53; P<0.0001), and noncalcified plaque volumes (ß, -0.08 [95% CI, -0.10 to -0.06] R2, 0.41; P<0.0001). Treatment of inflammatory stimulated endothelial cells with plasma with high Netrin-1 level resulted in reduced endothelial inflammation and consequently, less monocyte adhesion. CONCLUSIONS: Netrin-1 plasma levels are lower in patients with subclinical atherosclerosis and in patients with arterial wall inflammation. Netrin-1 is not associated with plaque vulnerability; however, it is negatively correlated to plaque burden, suggesting that Netrin-1 is involved in some, but not all, stages of atherosclerosis.

Understanding netrins and semaphorins in mature endothelial cell biology.

Netrins and semaphorins are known as neuronal guidance molecules that are important to the facilitate patterning of the nervous system in embryonic development. In recent years, their function has been broadened to guide development in other systems, including the vascular system, where netrins and semaphorins critically contribute to the development of the vascular system. Evidence is accumulating that these guidance cues are also of critical importance in the biology of the mature endothelium by regulating the maintenance of endothelial quiescence. Here we review our current insights into the roles of netrins and semaphorins in endothelial cell survival, self-renewing, barrier function, response to wall shear stress, and control of the vascular tone. We also provide suggestions for future research into the functions of netrins and semaphorins in mature endothelial cell biology.

Molecular basis of familial hypercholesterolemia.

PURPOSE OF REVIEW: To provide an overview about the molecular basis of familial hypercholesterolemia. RECENT FINDINGS: Familial hypercholesterolemia is a common hereditary cause of premature coronary heart disease. It has been estimated that 1 in every 250 individuals has heterozygous familial hypercholesterolemia and that fewer than 1% of patients with familial hypercholesterolemia have been identified across the globe. If heterozygous familial hypercholesterolemia is left untreated, it is likely that coronary heart disease will manifest clinically prior to the age of 55 years and that half of all patients will prematurely die from the consequences of myocardial infarction. It is crucial to understand the molecular basis of familial hypercholesterolemia to diagnose familial hypercholesterolemia properly. SUMMARY: The phenotype of familial hypercholesterolemia is caused by more than 1700 mutations the LDLR, apoB and PCSK9 genes, which explains approximately 85% of familial hypercholesterolemia cases. By means of next-generation sequencing, an increasing number of mutations in established and putative novel genes associated with this phenotype have been identified.

New Drugs for Atherosclerosis.

Atherosclerosis, the underlying process that ultimately leads to clinical cardiovascular disease (CVD), is caused by the multifactorial interaction of various conditions, and dyslipidemia is widely acknowledged as 1 of the crucial risk factors in this process. Statin drugs have been shown to decrease low-density lipoprotein cholesterol and CVD morbidity as well as mortality and are therefore pivotal in CVD prevention. Despite the use of statin drugs, CVD remains a leading cause of mortality worldwide, which suggests that additional lipid-lowering therapies are warranted. Several novel therapeutic agents, which are described in this review, are now well on their way in their respective development paths and might revolutionize anti-atherosclerotic drug therapy.