**5. Lp(a): A causal risk factor for atherosclerosis, CHD and stroke**

In MedLine and other databanks, there are more than 1500 papers listed dealing with this topic. Thus, it is almost impossible to consider all these publications in this report. Therefore, in this article, we concentrate mainly on the most recent findings; this does not imply that older references might be of lesser relevance. Semiquantitative measurements of Lp(a) in a Scandi‐ navian collective by the "father" of Lp(a), Kare Berg, revealed that individuals with a more pronounced "sinking pre-β band" [= Lp(a)] is found in lipoprotein electrophoresis correlated with the appearance of angina pectoris and CHD [14]. The first quantitative measurements of Lp(a) were in fact carried out by rocket electrophoresis in our laboratory in cooperation with P. Avogaro from Venice. In that case-control study, where 183 probands were included, it was found that the relative risk (RR) of suffering from myocardial infarction (MI)—depending on the applied cutoff value—was approximately 2-fold higher than in healthy controls [15]. This led to the adoption of an upper cutoff value of 30 mg/dl in most subsequent studies. In our first publication, we also could show that patients with type-IIa hyperlipoproteinemia (familial hypercholesterolemia, FH) exhibited a 6-fold higher risk of myocardial infarction (MI). Most of the subsequent studies that were published from various laboratories confirmed a positive correlation of Lp(a) plasma levels with CHD and MI. Some of the studies in fact were also negative, i.e. no relation of Lp(a) with atherosclerotic diseases could be established (for a review, see ref. [16]). A stab in the back to Lp(a) research in fact was given in 1993 by the article from Ridker et al. [17], who could not find any significant relation between Lp(a) and the risk for CHD in a nested case-control evaluation in the Physician's Health Study with almost 15,000 probands "In this prospective study of predominantly middle-aged white men, we found no evidence of association between Lp(a) level and risk of future MI. These data do not support the use of Lp(a) level as a screening tool to define cardiovascular risk among this population." These findings of Ridker et al [17] might have been based on the fact that the methodology used for Lp(a) quantification was subject to criticism.

Some years later, Lp(a) research encountered a revival after the publication of new data from several research groups in 2009–2011. These studies comprised >100.000 patients or probands and, for the first time, revealed beyond any doubt a significant causal relationship between elevated plasma Lp(a) and CHD ([2–4, 18,19]). Of note are studies from the last 3 years which underline the significance of Lp(a) as a risk factor for atherosclerotic cardiovascular diseases:

Significant amounts of radioactivity from labeled Lp(a), however, were also found in kidney, spleen, lung and pancreas, yet it is unknown whether these organs are of relevance for Lp(a) catabolism in humans. Since the liver is the principal organ for the LDL-receptor-mediated catabolism of apoB-containing lipoproteins, it was of interest to study this particular pathway for Lp(a) catabolism. *In vivo* studies carried out in our laboratory as well as by other groups, however, revealed that Lp(a) only has a low affinity to the LDL-R. The main argument for this allegation is the fact that Lp(a) is catabolized in homozygous FH patients with the same rate as compared to healthy control individuals [12]. Since pathways involved in Lp(a) catabolism —and in particular the role of specific receptors—is of eminent importance for strategies to develop Lp(a)-lowering drugs, many attempts have been made to identify binding proteins (receptors) that might be specific for Lp(a). Actually there is hardly any lipoprotein receptor that had not been found to bind Lp(a), including LRP, VLDL-R, asialo-glycoprotein receptor, different scavenger receptors and others. Unfortunately, all these results are based on *in vitro* studies that may have little relevance for the *in vivo* situation. One pathway that appears to be a hot candidate for Lp(a) catabolism is the binding of apo(a) kringle to lysine (Lys)-rich cell surface proteins. Along these lines, we actually demonstrated in previous experiments that

Graz, Austria).

140 Lipoproteins - From Bench to Bedside

**Figure 1.** Inhibition of apo(a) transcription by bile acids. Chenodeoxycholic acid (CDCA), the FXR agonist with the highest affinity in humans, binds and activates FXR leading to a displacement of that complex from the cytoplasm to the nucleus. The complex interferes with HNF4α binding to DR-1, a key response element in the apo(a) promoter and in turn silences apo(a) transcription. With permission of the Medical University of Graz (copyrights held by the MUG

> The PROCARDIS Consortium asked the question that had been discussed for a long time, whether different apo(a) isoforms with different number of K-IV repeats would exert differ‐ ences in their atherogenicity [20]. There were actually indications in the literature that not only the actual plasma concentration of Lp(a) but also the size polymorphism may reflect the risk of atherosclerosis. Thus, in the PROCARDIS study, including some 1000 patients and a similar

number of control individuals, plasma Lp(a) concentrations were measured by latex-enhanced immune-turbidimetry (see below) and apo(a) isoforms were assayed by SDS-polyacrylamide gel electrophoresis followed by immune blotting, using the isoform-standard from Immuno A.G., Vienna. Unfortunately, Immuno A.G. does not exist anymore and isoform standards are nowadays hard to obtain. The authors of PROCARDIS calculated the odds ratio (OR) of patients and controls between the first and last quintile before and after adjusting for the number of K-IV repeats. In both calculations, an OR of 2.05 (*p* < 0.001) was found, i.e. no difference could be observed whether the apo(a) size polymorphism was taken into consid‐ eration or not. This report appears to quite definitely conclude this debate and is proof that Lp(a) exerts its atherogenicity through its plasma concentration and not through possible structural differences in K-IV repeats. In an editorial to this report, F. Kronenberg (Innsbruck) pointed out that the analysis of SNPs—in particular rs41272114, rs10455872 and rs3798220, which exhibit the strongest association to plasma Lp(a) concentrations can neither be taken as surrogates nor as substitutions for the number of K-IV repeats. He further pointed out that more than half the number of individuals with isoforms containing less than 22 K-IV repeats are not recorded by this SNP analysis mentioned above.

In a further publication by the PROCARDIS Consortium published in ATVB [21], the question was asked as to what extent the LPA "null allele" (rs41272114) might influence the plasma concentration of Lp(a) in heterozygous individuals and if it might be a determinant for atherogenic risk. In this study comprising some 8000 CAD patients, an allele frequency for rs41272114 of approximately 3% was found. Patients containing the null allele exhibited significantly lower plasma Lp(a) levels as compared to control individuals without the rs41272114 allele (OR 0.79; *p* = 0.023). According to findings from the group of G. Utermann [22], the rs41272114 SNP represents a donor-splice site mutation leading to the biosynthesis of a truncated apo(a) with only 7 K-IVs (K-IV 1–7) in total and no K-V or protease domain. As a consequence of the absence of K-IV type 9, which contains the only free –SH group in apo(a) and is responsible for the covalent binding to apoB-100, the truncated apo(a) fragments are well secreted from the liver into the blood but do not assemble with LDL and thus are rapidly degraded and removed from the circulation. The PROCARDIS study also proved that individuals with only one apo(a) isoform exhibit a large variation in their plasma Lp(a) concentrations and that there exists a sigmoid correlation between the number of K-IV repeats and plasma Lp(a) levels. The question of the mechanism that causes this variation, however, could not be answered by this study. The authors of the PROCARDIS Consortium claimed, on the basis of their results, that in future epidemiological studies by SNP analysis for the assessment of the CAD risk, the rs41272114 polymorphism must be taken into consideration as a matter of state-of-the-art experiments.

Further support of the hypothesis published in 1981 by our group [15] indicating that Lp(a) might be a significant risk factor for MI comes from the "Bruneck Study" comprising 826 male and female probands [23]. In a recall survey after 15 years, it was found that the inclusion of Lp(a) in the Framingham algorithm for the risk assessment of CHD, an improvement of 0.016 in the C-index was reached. Consideration of Lp(a) plasma levels improved the hit rate in the prediction of CHD by 40%.
