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Abstract The risk of atherosclerotic cardiovascular diseases (ASCVDs) can be reduced by lowering low-density lipoprotein cholesterol (LDL-C) concentrations. Nevertheless, ASCVDs still cause most deaths worldwide. Here, we discuss the prevention of ASCVD and the event risk with a focus on heart-healthy diets, i.e., low intakes of saturated and trans-fatty acids and cholesterol, and high intakes of unsaturated fatty acids, viscous fibre, and dietary phytostanols as fatty acid esters, according to international dyslipidaemia treatment guidelines. Calculations based on both FINRISK and Cholesterol Treatment Trialists’ Collaborators regression equations indicate that heart-healthy diets combined with phytostanol ester reduce LDL-C concentrations to such an extent that the 10-year estimated reduction in the incidence of coronary artery disease would be 23%. This information can be used, in particular, to prevent the development of subclinical atherosclerosis in healthy middle-aged populations and the progression of atherosclerosis to ASCVD. The outcome of simple and feasible dietary changes, and, when needed, combined with statins, can be significant: reduced mortality, an increased number of healthy life-years, and reduced healthcare costs.

There is growing concern that the severe respiratory disease in birds (avian influenza or ‘bird flu’) caused by the H5N1 influenza virus, might potentially spread more widely to humans and cause a pandemic. Here we discuss clinical issues related to human infections by the highly pathogenic H5N1 subtype of the avian influenza A virus and make a clinical comparison with recent information obtained from studies of SARS-CoV-2 infection. Firstly, we consider the potential increase in cardiovascular events in humans infected with the H5N1 virus. Like SARS-CoV-2 infection, H5N1 infection may result in endothelial dysfunction and the associated procoagulant and prothrombotic state, and via this mechanism, the infection can potentially increase cardiovascular morbidity, especially in vulnerable individuals with pre-existing cardiovascular disease. Secondly, we discuss the potential beneficial role of statin use, both in the prophylaxis and the treatment of individuals with influenza A(H5N1), as was found favorable for the treatment of COVID-19 caused by SARS-CoV-2 infection.

In this short narrative review, we aim at defining the pathophysiological role endothelial dysfunction in the observed COVID-19–associated rise in risk of cardiovascular disease. Variants of the SARS-CoV-2 virus have caused several epidemic waves of COVID-19, and the emergence and rapid spread of new variants and subvariants are likely. Based on a large cohort study, the incidence rate of SARS-CoV-2 reinfection is about 0.66 per 10 000 person-weeks. Both the first infection and reinfection with SARS-CoV-2 increase cardiac event risk, particularly in vulnerable patients with cardiovascular risk factors and the accompanying systemic endothelial dysfunction. By worsening pre-existing endothelial dysfunction, both the first infection and reinfection with ensuing COVID-19 may turn the endothelium procoagulative and prothrombotic, and ultimately lead to local thrombus formation. When occurring in an epicardial coronary artery, the risk of an acute coronary syndrome increases, and when occurring in intramyocardial microvessels, scattered myocardial injuries will ensue, both predisposing the COVID-19 patients to adverse cardiovascular outcomes. In conclusion, considering weakened protection against the cardiovascular risk-enhancing reinfections with emerging new subvariants of SARS-CoV-2, treatment of COVID-19 patients with statins during the illness and thereafter is recommended, partly because the statins tend to reduce endothelial dysfunction.

AbstractPatients with hypercholesterolemia often have coronary microvascular dysfunction (CMD). Viral infections, such as the SARS-CoV-2 infection, may also result in CMD. Three non-randomized studies have shown significant beneficial effects of statins on CMD in non-infected patients. Similarly, in SARS-CoV-2 - infected patients one beneficial mechanism of action of statins may be the amelioration of endothelial dysfunction, which is a major driver of CMD. Apart from statins, lipoprotein apheresis and PCSK9 inhibitors can also improve or even reverse CMD. The potential reversal of CMD by using effective cholesterol-lowering medications during and after COVID-19 infection, especially in hypercholesterolemic COVID-19 patients, is important.KEY MESSAGESCoronary microvascular dysfunction (CMD) is common in patients hospitalized with SARS-CoV-2 infectionThree nonrandomized studies in non-infected patients are showing the beneficial effects of statin treatment on CMDEffective cholesterol-lowering medication during and after SARS-CoV-2 infection, especially in hypercholesterolemic COVID-19 patients, is of great significance

Based on separate protective mechanisms related to lipid metabolism, viral cell entry and inflammation, fibrate treatment might be advantageous among patients who have been taking fibrates before SARS-CoV-2 infection and continue taking them during the infection. Based on published data on hospitalized COVID-19 patients, we recommend that the clinicians should ask their patients with metabolic syndrome who are already taking fibrates to continue fibrate treatment during the COVID-19 illness. This recommendation applies to both outpatients and hospitalized patients. However, results from the ongoing randomized controlled trials (RCTs) using fenofibrate treatment for the prevention or treatment of COVID-19 have yet to prove that fenofibrate is clinically significant for this indication.KEY MESSAGESThe role of fibrates as a repurpose to treat SARS-CoV-2 is under investigation in at least three ongoing RCTs.Obesity, diabetes, hypertension and dyslipidaemia, individually or clustered as a discrete phenotype, the metabolic syndrome, typically associate with a more severe course of COVID-19.Fibrate treatment seems to be most advantageous among patients who have been taken fibrates before SARS-CoV-2 infection and are continuing to take them during the infection.We recommend that the clinicians encourage their patients who are already taking fibrate to continue using the drug throughout the COVID-19 illness.

Abstract Saccular intracranial aneurysm (sIA) rupture leads to subarachnoid haemorrhage and is preceded by chronic inflammation and atherosclerotic changes of the sIA wall. Increased lymphangiogenesis has been detected in atherosclerotic extracranial arteries and in abdominal aortic aneurysms, but the presence of lymphatic vessels in sIAs has remained unexplored. Here we studied the presence of lymphatic vessels in 36 intraoperatively resected sIAs (16 unruptured and 20 ruptured), using immunohistochemical and immunofluorescence stainings for lymphatic endothelial cell (LEC) markers. Of these LEC-markers, both extracellular and intracellular LYVE-1-, podoplanin-, VEGFR-3-, and Prox1-positive stainings were detected in 83%, 94%, 100%, and 72% of the 36 sIA walls, respectively. Lymphatic vessels were identified as ring-shaped structures positive for one or more of the LEC markers. Of the sIAs, 78% contained lymphatic vessels positive for at least one LEC marker. The presence of LECs and lymphatic vessels were associated with the number of CD68+ and CD163+ cells in the sIA walls, and with the expression of inflammation indicators such as serum amyloid A, myeloperoxidase, and cyclo-oxygenase 2, with the presence of a thrombus, and with the sIA wall rupture. Large areas of VEGFR-3 and α-smooth muscle actin (αSMA) double-positive cells were detected in medial parts of the sIA walls. Also, a few podoplanin and αSMA double-positive cells were discovered. In addition, LYVE-1 and CD68 double-positive cells were detected in the sIA walls and in the thrombus revealing that certain CD68+ macrophages are capable of expressing LEC markers. This study demonstrates for the first time the presence of lymphatic vessels in human sIA walls. Further studies are needed to understand the role of lymphatic vessels in the pathogenesis of sIA.

IntroductionLipoprotein(a) (Lp(a)) is an LDL-like particle with an additional apolipoprotein (apo)(a) covalently attached. Elevated levels of circulating Lp(a) are a risk factor for atherosclerosis. A proinflammatory role for Lp(a) has been proposed, but its molecular details are incompletely defined.Methods and resultsTo explore the effect of Lp(a) on human macrophages we performed RNA sequencing on THP-1 macrophages treated with Lp(a) or recombinant apo(a), which showed that especially Lp(a) induces potent inflammatory responses. Thus, we stimulated THP-1 macrophages with serum containing various Lp(a) levels to investigate their correlations with cytokines highlighted by the RNAseq, showing significant correlations with caspase-1 activity and secretion of IL-1β and IL-18. We further isolated both Lp(a) and LDL particles from three donors and then compared their atheroinflammatory potentials together with recombinant apo(a) in primary and THP-1 derived macrophages. Compared with LDL, Lp(a) induced a robust and dose-dependent caspase-1 activation and release of IL-1β and IL-18 in both macrophage types. Recombinant apo(a) strongly induced caspase-1 activation and IL-1β release in THP-1 macrophages but yielded weak responses in primary macrophages. Structural analysis of these particles revealed that the Lp(a) proteome was enriched in proteins associated with complement activation and coagulation, and its lipidome was relatively deficient in polyunsaturated fatty acids and had a high n-6/n-3 ratio promoting inflammation.DiscussionOur data show that Lp(a) particles induce the expression of inflammatory genes, and Lp(a) and to a lesser extent apo(a) induce caspase-1 activation and IL-1 signaling. Major differences in the molecular profiles between Lp(a) and LDL contribute to Lp(a) being more atheroinflammatory.

SARS-CoV-2 infection continues to cause increased morbidity and mortality, and due to the slow pace of vaccination COVID-19 will probably remain a global burden to health systems for a long time. Unfortunately, the necessary prevention and treatment strategies of COVID-19 have led to restriction measures that are hampering the routine care of common chronic metabolic conditions like hypercholesterolemia. It is of particular concern that during the acute phase of COVID-19, the control of pre-existing metabolic diseases tends to get worse which again increases the risk for complications and a poor outcome in these patients. A significant contributor to these complications is endothelial dysfunction which is associated with COVID-19. This Commentary will discuss the impact of COVID-19 on endothelial function particularly in patients with familial hypercholesterolemia (FH), a metabolic inherited disease known to in itself adversely affect endothelial function. There should be no hesitation to continue with statin therapy in severe hypercholesterolemic patients with COVID-19. We argue that in FH patients with COVID-19 the clinicians need even consider intensifying statin therapy as well as the addition of other lipid-lowering agents, such as proprotein convertase subtilisin/kexin type 9(PCSK9) inhibitors. In contrast to statins, the PCSK9 inhibitors lower lipoprotein(a) [Lp(a)] level, and, accordingly, these latter drugs need to be considered particularly in FH patients with an elevated level of Lp(a). This call applies to the in-hospital stay and also beyond. When considering that the vasculopathic effects of COVID-19 may persist, a long-term follow-up of individualized therapies in FH patients is warranted.

ObjectivesImpaired protein kinase signaling is a hallmark of ischemic heart disease (IHD). Inadequate understanding of the pathological mechanisms limits the development of therapeutic approaches. We aimed to identify the key cardiac kinases and signaling pathways in patients with IHD with an effort to discover potential therapeutic strategies.MethodsCardiac kinase activity in IHD left ventricle (LV) and the related signaling pathways were investigated by kinomics, transcriptomics, proteomics, and integrated multi-omics approach.ResultsProtein kinase A (PKA) and protein kinase G (PKG) ranked on top in the activity shift among the cardiac kinases. In the IHD LVs, PKA activity decreased markedly compared with that of controls (62% reduction, p = 0.0034), whereas PKG activity remained stable, although the amount of PKG protein increased remarkably (65%, p = 0.003). mRNA levels of adenylate cyclases (ADCY 1, 3, 5, 9) and cAMP-hydrolysing phosphodiesterases (PDE4A, PDE4D) decreased significantly, although no statistically significant alterations were observed in that of PKGs (PRKG1 and PRKG2) and guanylate cyclases (GUCYs). The gene expression of natriuretic peptide CNP decreased remarkably, whereas those of BNP, ANP, and neprilysin increased significantly in the IHD LVs. Proteomics analysis revealed a significant reduction in protein levels of “Energy metabolism” and “Muscle contraction” in the patients. Multi-omics integration highlighted intracellular signaling by second messengers as the top enriched Reactome pathway.ConclusionThe deficiency in cAMP/PKA signaling pathway is strongly implicated in the pathogenesis of IHD. Natriuretic peptide CNP could be a potential therapeutic target for the modulation of cGMP/PKG signaling.

Abstract In atherosclerotic lesions, blood-derived monocytes differentiate into distinct macrophage subpopulations, and further into cholesterol-filled foam cells under a complex milieu of cytokines, which also contains macrophage-colony stimulating factor (M-CSF) and granulocyte–macrophage-colony stimulating factor (GM-CSF). Here we generated human macrophages in the presence of either M-CSF or GM-CSF to obtain M-MØ and GM-MØ, respectively. The macrophages were converted into cholesterol-loaded foam cells by incubating them with acetyl-LDL, and their atheroinflammatory gene expression profiles were then assessed. Compared with GM-MØ, the M-MØ expressed higher levels of CD36, SRA1, and ACAT1, and also exhibited a greater ability to take up acetyl-LDL, esterify cholesterol, and become converted to foam cells. M-MØ foam cells expressed higher levels of ABCA1 and ABCG1, and, correspondingly, exhibited higher rates of cholesterol efflux to apoA-I and HDL2. Cholesterol loading of M-MØ strongly suppressed the high baseline expression of CCL2, whereas in GM-MØ the low baseline expression CCL2 remained unchanged during cholesterol loading. The expression of TNFA, IL1B, and CXCL8 were reduced in LPS-activated macrophage foam cells of either subtype. In summary, cholesterol loading converged the CSF-dependent expression of key genes related to intracellular cholesterol balance and inflammation. These findings suggest that transformation of CSF-polarized macrophages into foam cells may reduce their atheroinflammatory potential in atherogenesis.

Circulating apolipoprotein B-containing lipoproteins, notably the low-density lipoproteins, enter the inner layer of the arterial wall, the intima, where a fraction of them is retained and modified by proteases, lipases, and oxidizing agents and enzymes. The modified lipoproteins and various modification products, such as fatty acids, ceramides, lysophospholipids, and oxidized lipids induce inflammatory reactions in the macrophages and the covering endothelial cells, initiating an increased leukocyte diapedesis. Lipolysis of the lipoproteins also induces the formation of cholesterol crystals with strong proinflammatory properties. Modified and aggregated lipoproteins, cholesterol crystals, and lipoproteins isolated from human atherosclerotic lesions, all can activate macrophages and thereby induce the secretion of proinflammatory cytokines, chemokines, and enzymes. The extent of lipoprotein retention, modification, and aggregation have been shown to depend largely on differences in the composition of the circulating lipoprotein particles. These properties can be modified by pharmacological means, and thereby provide opportunities for clinical interventions regarding the prevention and treatment of atherosclerotic vascular diseases.

Atherosclerotic arterial plaques and malignant solid tumors contain macrophages, which participate in anaerobic metabolism, acidosis, and inflammatory processes inherent in the development of either disease. The tissue-resident macrophage populations originate from precursor cells derived from the yolk sac and from circulating bone marrow-derived monocytes. In the tissues, they differentiate into varying functional phenotypes in response to local microenvironmental stimulation. Broadly categorized, the macrophages are activated to polarize into proinflammatory M1 and anti-inflammatory M2 phenotypes; yet, noticeable plasticity allows them to dynamically shift between several distinct functional subtypes. In atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates within macrophages as cytoplasmic lipid droplets thereby generating macrophage foam cells, which are involved in all steps of atherosclerosis. The conversion of macrophages into foam cells may suppress the expression of given proinflammatory genes and thereby initiate their transcriptional reprogramming toward an anti-inflammatory phenotype. In this particular sense, foam cell formation can be considered anti-atherogenic. The tumor-associated macrophages (TAMs) may become polarized into anti-tumoral M1 and pro-tumoral M2 phenotypes. Mechanistically, the TAMs can regulate the survival and proliferation of the surrounding cancer cells and participate in various aspects of tumor formation, progression, and metastasis. The TAMs may accumulate lipids, but their type and their specific roles in tumorigenesis are still poorly understood. Here, we discuss how the phenotypic and functional plasticity of macrophages allows their multifunctional response to the distinct microenvironments in developing atherosclerotic lesions and in developing malignant tumors. We also discuss how the inflammatory reactions of the macrophages may influence the development of atherosclerotic plaques and malignant tumors, and highlight the potential therapeutic effects of targeting lipid-laden macrophages in either disease.

Heterozygous familial hypercholesterolemia (HeFH) patients are the prime example of subjects who are at high risk for both acute myocardial infarction (AMI) and ischemic stroke during, and post, SARS-CoV-2 infection. HeFH per se, if left untreated, results in premature clinical atherosclerosis often presenting in the fourth or fifth decade of life. The other concern in HeFH is endothelial dysfunction which is already evident from early childhood. In untreated HeFH patients, the severe hypercholesterolemia causes endothelial dysfunction from an early age, and as a result thereof, atherosclerotic lesions develop prematurely, particularly in the coronary arteries, and result in further endothelial dysfunction and inflammation in these critical segments of the arterial tree. As the pre-existing endothelial dysfunction in HeFH patients is most likely sensitive to further direct and indirect SARS-CoV-2 virus-dependent damage, we can infer that HeFH serves as an example of a comorbidity that predicts a poorer prognosis with COVID-19 infection. Indeed, a large US national database study showed that patients diagnosed with HeFH and SARS-CoV-2 infection had significantly increased Annualized Incidence Density Rates (AIDRs) of AMI when compared to matched HeFH controls not having been diagnosed with SARS-CoV-2 infection. Effective cholesterol lowering is essential for the prevention, or at least alleviation, of the detrimental effects of SARS-CoV-2 infection among HeFH patients. Due to the pre-existing subclinical or even clinical atherosclerotic cardiovascular disease in subjects with HeFH, cholesterol-lowering treatment needs to be continued or, better still, intensified during, and for an extended period post, SARS-CoV-2 infection.

Background: Long-term treatment with the vitamin K antagonist warfarin is widely used for the prevention of venous thrombosis and thromboembolism. However, vitamin K antagonists may promote arterial calcification, a phenomenon that has been previously studied in coronary and peripheral arteries, but not in extracranial carotid arteries. In this observational cohort study, we investigated whether warfarin treatment is associated with calcification of atherosclerotic carotid arteries.Methods: Overall, 500 consecutive patients underwent carotid endarterectomy, 82 of whom had received long-term warfarin therapy. The extent of calcification was assessed with preoperative computed tomography angiography, and both macroscopic morphological grading and microscopic histological examination of each excised carotid plaque were performed after carotid endarterectomy.Results: Compared with non-users, warfarin users had significantly more computed tomography angiography-detectable vascular calcification in the common carotid arteries (odds ratio 2.64, 95% confidence interval 1.51–4.63, P < 0.001) and even more calcification in the internal carotid arteries near the bifurcation (odds ratio 18.27, 95% confidence interval 2.53–2323, P < 0.001). Histological analysis revealed that the intramural calcified area in plaques from warfarin users was significantly larger than in plaques from non-users (95% confidence interval 3.36–13.56, P = 0.0018).Conclusions: Long-lasting warfarin anticoagulation associated with increased calcification of carotid atherosclerotic plaques, particularly in locations known to be the predilection sites of stroke-causing plaques. The clinical significance of this novel finding warrants further investigations.

There is a continuing need for research about the underlying mechanisms behind ischemic strokes in COVID-19 patients. Pre-existing endothelial dysfunction, especially if it is accompanied by a viral infection of the endothelial cells may present an important mechanism behind the immunothrombotic/thromboembolic complications of the COVID-19 illness. Here we emphasize that pharmacotherapy with statins could partly counteract such pathophysiological scenarios. Accordingly, using familial hypercholesterolemia (FH) as a pertinent example of a lifelong endothelial dysfunction, we aim to make the clinicians and consulting neurologists aware of statins as a possible adjuvant therapy in the context of an increased risk of ischemic stroke in patients with COVID-19. Based on recent clinical evidence, there is a need to encourage clinicians and consulting neurologists to continue or initiate effective statin treatment to prevent an ischemic stroke, particularly when they encounter a hypercholesterolemic COVID-19 patient with FH.

Abstract Background The focus of studies on high-density lipoproteins (HDL) has shifted from HDL-cholesterol (HDL-C) to HDL function. We recently demonstrated that low USF1 expression in mice and humans associates with high plasma HDL-C and low triglyceride levels, as well as protection against obesity, insulin resistance, and atherosclerosis. Here, we studied the impact of USF1 deficiency on HDL functional capacity and macrophage atherogenic functions, including inflammation, cholesterol efflux, and cholesterol accumulation. Methods We used a congenic Usf1 deficient mice in C57Bl/6JRccHsd background and blood samples were collected to isolate HDL for structural and functional studies. Lentiviral preparations containing the USF1 silencing shRNA expression vector were used to silence USF1 in human THP-1 and Huh-7 cells. Cholesterol efflux from acetyl-LDL loaded THP-1 macrophages was measured using HDL and plasma as acceptors. Gene expression analysis from USF1 silenced peritoneal macrophages was carried out using Affymetrix protocols. Results We show that Usf1 deficiency not only increases HDL-C levels in vivo, consistent with elevated ABCA1 protein expression in hepatic cell lines, but also improves the functional capacity of HDL particles. HDL particles derived from Usf1 deficient mice remove cholesterol more efficiently from macrophages, attributed to their higher contents of phospholipids. Furthermore, silencing of USF1 in macrophages enhanced the cholesterol efflux capacity of these cells. These findings are consistent with reduced inflammatory burden of USF1 deficient macrophages, manifested by reduced secretion of pro-inflammatory cytokines MCP-1 and IL-1β and protection against inflammation-induced macrophage cholesterol accumulation in a cell-autonomous manner. Conclusions Our findings identify USF1 as a novel factor regulating HDL functionality, showing that USF1 inactivation boosts cholesterol efflux, reduces macrophage inflammation and attenuates macrophage cholesterol accumulation, linking improved macrophage cholesterol metabolism and inflammatory pathways to the antiatherogenic function of USF1 deficiency.

Despite the increased frequency and scale of wildfire-related catastrophes, there has been little or no effective and coordinated international policy to address their highly negative impact. Possibly a generalized approach to respond to such major events could be modeled on existing international safety investigation policies and agreements that already have proved successful. The International Civil Aviation Organization (ICAO) outlines safety investigations after international fatal aviation accidents. Although this well-established safety investigation protocol cannot be directly applied in acute wildfire-related accidents, it can offer a useful framework for establishing international guidelines to reduce risk of future wildfire catastrophes. The co-operation between safety investigation authorities has been shown to be fruitful especially for those less developed countries that have limited resources and experience related to accident investigations. While primarily an adaptive measure that can set practices to reduce vulnerability and fragility of ecosystems and human societies, the same policies could be seen as a climate change mitigation measure, as wildfires can contribute significantly to global CO2 emissions. Finally, the concept of independent and qualified safety investigations represents the principle of serendipity: disclosing by accident something that has not been foreseen. Feedback from reality compensates assumptions and limitations of feedforward analysis of complex systems that can only reveal their dynamics and performance in reality and over time.

ApoA-I, the main structural and functional protein of HDL particles, is cardioprotective, but also highly sensitive to proteolytic cleavage. Here, we investigated the effect of cardiac mast cell activation and ensuing chymase secretion on apoA-I degradation using isolated rat hearts in the Langendorff perfusion system. Cardiac mast cells were activated by injection of compound 48/80 into the coronary circulation or by low-flow myocardial ischemia, after which lipid-free apoA-I was injected and collected in the coronary effluent for cleavage analysis. Mast cell activation by 48/80 resulted in apoA-I cleavage at sites Tyr192 and Phe229, but hypoxic activation at Tyr192 only. In vitro, the proteolytic end-product of apoA-I with either rat or human chymase was the Tyr192-truncated fragment. This fragment, when compared with intact apoA-I, showed reduced ability to promote migration of cultured human coronary artery endothelial cells in a wound-healing assay. We propose that C-terminal truncation of apoA-I by chymase released from cardiac mast cells during ischemia impairs the ability of apoA-I to heal damaged endothelium in the ischemic myocardium.

Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.

Atherosclerotic lesions are often hypoxic and exhibit elevated lactate concentrations and local acidification of the extracellular fluids. The acidification may be a consequence of the abundant accumulation of lipid-scavenging macrophages in the lesions. Activated macrophages have a very high energy demand and they preferentially use glycolysis for ATP synthesis even under normoxic conditions, resulting in enhanced local generation and secretion of lactate and protons. In this review, we summarize our current understanding of the effects of acidic extracellular pH on three key players in atherogenesis: macrophages, apoB-containing lipoproteins, and HDL particles. Acidic extracellular pH enhances receptor-mediated phagocytosis and antigen presentation by macrophages and, importantly, triggers the secretion of proinflammatory cytokines from macrophages through activation of the inflammasome pathway. Acidity enhances the proteolytic, lipolytic, and oxidative modifications of LDL and other apoB-containing lipoproteins, and strongly increases their affinity for proteoglycans, and may thus have major effects on their retention and the ensuing cellular responses in the arterial intima. Finally, the decrease in the expression of ABCA1 at acidic pH may compromise cholesterol clearance from atherosclerotic lesions. Taken together, acidic extracellular pH amplifies the proatherogenic and proinflammatory processes involved in atherogenesis.

Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [3H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [3H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A2-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.

HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL2 and HDL3 subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL3 than in HDL2. Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects.

During atherogenesis, the extracellular pH of atherosclerotic lesions decreases. Here, we examined the effect of low, but physiologically plausible pH on aggregation of modified LDL, one of the key processes in atherogenesis. LDL was treated with SMase, and aggregation of the SMase-treated LDL was followed at pH 5.5–7.5. The lower the pH, the more extensive was the aggregation of identically prelipolyzed LDL particles. At pH 5.5–6.0, the aggregates were much larger (size >1 µm) than those formed at neutral pH (100–200 nm). SMase treatment was found to lead to a dramatic decrease in α-helix and concomitant increase in β-sheet structures of apoB-100. Particle aggregation was caused by interactions between newly exposed segments of apoB-100. LDL-derived lipid microemulsions lacking apoB-100 failed to form large aggregates. SMase-induced LDL aggregation could be blocked by lowering the incubation temperature to 15°C, which also inhibited the changes in the conformation of apoB-100, by proteolytic degradation of apoB-100 after SMase-treatment, and by HDL particles. Taken together, sphingomyelin hydrolysis induces exposure of protease-sensitive sites of apoB-100, whose interactions govern subsequent particle aggregation. The supersized LDL aggregates may contribute to the retention of LDL lipids in acidic areas of atherosclerosis-susceptible sites in the arterial intima.

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34+ progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34+ progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A2 (sPLA2-IIa and sPLA2-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA2-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA2-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA2-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.

Electronegative LDL [LDL(−)] is an atherogenic subfraction of plasma LDL that has increased apolipoprotein E (apoE) and apoC-III content, high density, and increased susceptibility to aggregation. These characteristics suggest that LDL(−) could bind to proteoglycans (PGs); therefore, our aim was to evaluate its affinity to PGs. Binding of LDL(−) and native LDL [LDL(+)] to human aortic PGs was determined by precipitation of LDL-glycosaminoglycan complexes, LDL incubation in PG-coated microtiter wells, and affinity chromatography on PG column. All methods showed that LDL(−) had higher binding affinity to PGs than did LDL(+). PG capacity to bind LDL(−) was increased approximately 4-fold compared with LDL(+) in precipitation and microtiter assays. Chromatography on PG column showed LDL(−) to consist of two subpopulations, one with higher and one with lower PG binding affinity than LDL(+). Unexpectedly, the lower PG affinity subpopulation had increased apoE and apoC-III content. In contrast, the high PG affinity subpopulation presented phospholipase C (PLC)-like activity and increased aggregation. These results suggest that PLC-like activity could alter LDL lipid composition, thereby promoting particle aggregation and binding to PGs. This propensity of a subpopulation of LDL(−) to bind to PGs could facilitate its retention in the extracellular matrix of arterial intima and contribute to atherosclerosis progression.

The main antiatherogenic function of HDL is to promote the efflux of cholesterol from peripheral cells and transport it to the liver for excretion in a process termed reverse cholesterol transport. The aim of this study was to evaluate the cholesterol efflux capacity in low- and high-HDL subjects by utilizing monocytes and serum from 18 low-HDL and 15 high-HDL subjects. Low and high HDL levels were defined, respectively, as HDL ≤10th and HDL ≥90th Finnish age/sex-specific percentile. Cholesterol efflux from [3H]cholesterol-oleate-acetyl-LDL-loaded monocyte-derived macrophages to standard apolipoprotein A-I (apoA-I), HDL2, and serum was measured. In addition, cholesterol efflux from acetyl-LDL-loaded human THP-1 macrophages to individual sera (0.5%) derived from the study subjects was evaluated. Cholesterol efflux to apoA-I, HDL2, and serum from macrophage foam cells derived from low- and high-HDL subjects was similar. The relative ABCA1 and ABCG1 mRNA expression levels in unloaded macrophages, as well as their protein levels in loaded macrophage foam cells, were similar in the two study groups. Cholesterol efflux from THP-1 foam cells to serum recovered from high-HDL subjects was slightly higher than that to serum from low-HDL subjects (P = 0.046). Cholesterol efflux from THP-1 macrophages to serum from study subjects correlated with serum apoB (P = 0.033), apoA-I (P = 0.004), apoA-II (P < 0.0001), and the percentage of apoA-I present in the form of preβ-HDL (P = 0.0001). Our data reveal that macrophages isolated from either low- or high-HDL subjects display similar cholesterol efflux capacity to exogenous acceptors. However, sera from low-HDL subjects have poorer cholesterol acceptor ability as compared with sera from high-HDL subjects.

The pathogenesis of aortic valve stenosis (AS) is characterized by the accumulation of LDL-derived cholesterol in the diseased valves. Since LDL particles also contain plant sterols, we investigated whether plant sterols accumulate in aortic valve lesions. Serum samples were collected from 82 patients with severe AS and from 12 control subjects. Aortic valves were obtained from a subpopulation of 21 AS patients undergoing valve surgery and from 10 controls. Serum and valvular total cholesterol and noncholesterol sterols were measured by gas-liquid chromatography. Noncholesterol sterols, including both cholesterol precursors and sterols reflecting cholesterol absorption, were detected in serum samples and aortic valves. The higher the ratios to cholesterol of the cholesterol precursors and absorption markers in serum, the higher their ratios in the stenotic aortic valves (r = 0.74, P < 0.001 for lathosterol and r = 0.88, P < 0.001 for campesterol). The valvular ratio to cholesterol of lathosterol correlated negatively with the aortic valve area (r = −0.47, P = 0.045), suggesting attenuation of cholesterol synthesis with increasing severity of AS. The higher the absorption of cholesterol, the higher the plant sterol contents in stenotic aortic valves. These findings suggest that local accumulation of plant sterols and cholesterol precursors may participate in the pathobiology of aortic valve disease.

Human atherosclerotic intima contains mast cells that secrete the neutral protease chymase into the intimal fluid, which also contains HDL-modifying proteins, such as cholesteryl ester transfer protein (CETP), in addition to abundant amounts of nascent discoidal HDL particles. Here, we studied chymase-dependent degradation of a) CETP isolated from human plasma and b) CETP-HDL complexes as well as the functional consequences of such degradations. Incubation with chymase caused a rapid cleavage of CETP, yielding a specific proteolytic pattern with a concomitant reduction in its cholesteryl ester transfer activity. These chymase-dependent effects were attenuated after CETP was complexed with HDL. This attenuation was more effective when CETP was complexed with HDL3 and HDL2 than with discoidal reconstituted high density lipoprotein (rHDL). Conversely, rHDL, but not spherical HDLs, was protected in such CETP complexes against functional inactivation by chymase. Thus, in contrast to the complexes of CETP with spherical HDLs, the ability of the CETP-rHDL complexes to promote cholesterol efflux from macrophage foam cells remained unchanged, despite treatment with chymase. In summary, complexation of CETP and HDL modifies their resistance to proteolytic inactivation: spherical HDLs protect CETP, and CETP protects discoidal HDL. These results suggest that in inflamed atherosclerotic intima, CETP, via its complexation with HDL, has a novel protective role in early steps of reverse cholesterol transport.

HDL promotes cholesterol efflux from peripheral cells via ABCA1 in the first step of reverse cholesterol transport (RCT). We investigated whether the early steps of RCT were disturbed in subjects with familial low HDL and an increased risk for early atherosclerosis. Cholesterol efflux from monocyte-derived macrophages to lipid-free apolipoprotein A-I (apoA-I; %) was measured in 22 patients with familial low HDL without Tangier disease mutations and in 21 healthy controls. In addition, we defined the different alleles of ABCA1 using single-nucleotide polymorphism haplotypes and measured ABCA1 and ABCG1 mRNA transcript levels in cholesterol-loaded macrophages. Similar ABCA1-mediated cholesterol efflux levels were observed for macrophages derived from control subjects and from low-HDL subjects. However, when efflux of cholesterol was estimated as cholesterol efflux to apoA-I (%)/relative ABCA1 mRNA expression level, cholesterol removal was significantly (P = 0.001) lower in the low-HDL group. Cholesterol-loaded macrophages from low-HDL subjects showed significantly increased levels of ABCA1 mRNA but not of ABCG1 mRNA and were more often carriers of the rare ABCA1 alleles L158 and R219K. These results suggest that defective ABCA1 function in cholesterol-loaded macrophages is one potential contributor to the impaired RCT process and the increased coronary heart disease risk in subjects with familial low HDL.

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.

Mast cell chymase, a chymotrypsin-like neutral protease, can proteolyze HDL3. Here we studied the ability of rat and human chymase to proteolyze discoidal preβ-migrating reconstituted HDL particles (rHDLs) containing either apolipoprotein A-I (apoA-I) or apoA-II. Both chymases cleaved apoA-I in rHDL at identical sites, either at the N-terminus (Tyr18 or Phe33) or at the C-terminus (Phe225), so generating three major truncated polypeptides that remained bound to the rHDL. The cleavage sites were independent of the size of the rHDL particles, but small particles were more susceptible to degradation than bigger ones. Chymase-induced truncation of apoA-I yielded functionally compromised rHDL with reduced ability to promote cellular cholesterol efflux. In sharp contrast to apoA-I, apoA-II was resistant to degradation. However, when apoA-II was present in rHDL that also contained apoA-I, it was degraded by chymase.We conclude that chymase reduces the ability of apoA-I in discoidal rHDL particles to induce cholesterol efflux by cleaving off either its amino- or carboxy-terminal portion. This observation supports the concept that limited extracellular proteolysis of apoA-I is one pathophysiologic mechanism leading to the generation and maintenance of foam cells in atherosclerotic lesions.

Lipid droplets and membrane material are produced in the extracellular matrix of the arterial intima during atherogenesis. Both in vitro and in vivo experimentation suggests that fusion of modified LDL particles leads to formation of such lipid droplets. Here we applied proton NMR spectroscopy to probe surface phospholipids phosphatidylcholine (PC) and sphingomyelin (SM) of LDL particles during proteolytic degradation of apolipoprotein B-100 (apoB-100). Initiation of apoB-100 degradation was accompanied by the abruptly increased intensity of the choline -N(CH3)3 resonance of PC molecules, indicating disruption of their interactions with apoB-100. However, subsequent particle fusion was accompanied by a steady decrease in the intensity of the choline resonances of both PC and SM. Electron microscopy of the proteolyzed LDL revealed irregularly shaped multilamellar membranes attached to aggregates of fused particles. This suggests formation of membrane material with low hydration, in which some of the atomic motions are hindered. Characterization of the behavior of the surface lipids of LDL particles during apoB-100 degradation and other types of LDL modification will aid in understanding molecular mechanisms leading to fusion and generation of multilamellar membrane material in the arterial intima during atherogenesis.—Pentikäinen, M. O., M. T. Hyvönen, K. Öörni, T. Hevonoja, A. Korhonen, E. M. P. Lehtonen-Smeds, M. Ala-Korpela, and P. T. Kovanen. Altered phospholipid-apoB-100 interactions and generation of extra membrane material in proteolysis-induced fusion of LDL particles.

Initiation of atherosclerosis is characterized by accumulation of aggregates of small lipid droplets and vesicles in the extracellular matrix of the arterial intima. The droplets and vesicles have features that suggest that they are formed from modified plasma-derived low density lipoprotein (LDL) particles. A variety of hydrolytic enzymes and prooxidative agents that could lead to extracellular assembly of LDL-derived droplets and vesicles are present in the arterial intima. In fact, in vitro studies have demonstrated that extensive oxidation of LDL and treatment of LDL with either proteolytic or lipolytic enzymes will induce LDL aggregation and fusion and treatment of LDL with cholesterol esterase will cause formation of vesicles. Fusion of LDL particles proceeds faster in vitro when they are bound to components of the extracellular matrix derived from the arterial intima, such as proteoglycans, and, depending on the type of modification, the strength of binding of modified LDL to the matrix components may either increase or decrease. In the present article, we discuss molecular mechanisms that provide clues as to how aggregated lipid droplets and vesicles may be derived from modified LDL particles. We also describe how these modified forms of LDL, by means of their trapping to the extracellular matrix, may lead to extracellular lipid accumulation in the arterial intima.—Öörni, K., M. O. Pentikäinen, M. Ala-Korpela, and P. T. Kovanen. Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions. J. Lipid Res. 2000. 41: 1703–1714.

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL3 by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL3 revealed that proteolysis for up to 24 h did not alter the integrity of the α-migrating HDL, whereas a minor peak containing particles of smaller size with preβ mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the αHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL3 with chymase inhibited binding of Mab A-I-9 (specific for preβ1HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL3 to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL3 by chymase failed to reduce the ability of HDL3 to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation.This differential sensitivity of the two key functions of HDL3 to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for preβ1HDL to promote the high-affinity efflux of cellular cholesterol, but not for the α-migrating HDL particles to activate LCAT.

Recent evidence suggests that fusion of low density lipoprotein (LDL) particles is a key process in the initial accumulation of lipid in the arterial intima. In order to gain a better understanding of this early event in the development of atherosclerosis, it would thus be necessary to characterize the process of LDL fusion in detail. Such studies, however, pose severe methodological difficulties, such as differentiation of particle fusion from aggregation. In this paper we describe the use of novel methodology, based on 1H NMR spectroscopy, to study lipoprotein particle fusion. To test the methodology, we chose proteolytic fusion of LDL particles, an in vitro model that has been well characterized in our laboratory. The spectroscopic data suggested that proteolysis of LDL with α-chymotrypsin induced slow initiation of fusion, which was followed by particle fusion at an increased rate. Moreover, 1H NMR spectroscopic data on different kinds of LDL interactions, for example, when LDL formed aggregates with antibodies against human apolipoprotein B-100, were obtained and compared with the electron microscopic characteristics of these preparations. An important finding was that limited aggregation of LDL particles did not disturb the 1H NMR spectroscopic parameters used for the detection of particle fusion and preserved the physicochemical information on the particles. The 1H NMR methodology developed is sensitive to and specific for low density lipoprotein (LDL) fusion and may also allow for studies of the fate of LDL particles in other in vitro preparations that mimic the arterial interactions in vivo.—Ala-Korpela, M., M. O. Pentikäinen. A. Korhonen, T. Hevonoja, J. Lounila, and P. T. Kovanen. Detection of low density lipoprotein particle fusion by proton nuclear magnetic resonance spectroscopy.

Accumulating work in experimental animals suggests that bradykinin (BK) exerts cardioprotective effects via bradykinin type-2 receptors (BK-2Rs). In human end-stage heart failure, BK-2Rs are significantly downregulated by mechanisms that have remained elusive. Heart tissues from idiopathic dilated cardiomyopathy (IDC; 𝑛=7), coronary heart disease (CHD; 𝑛=6), and normal patients (𝑛=6) were analyzed by RT-PCR, SSCP, and Western blotting. In normal and IDC hearts, BK-2R expression increased with age, with a lower relative increase in IDC hearts. BK-2R mRNA and protein levels showed a positive linear correlation, suggesting transcriptional regulation. Two known BK-2R promoter polymorphisms, −58T/C and −9/+9, were found to be present in the study population. The allelic frequencies for the C-allele in −58T/C were 0.58 in normal and CHD hearts and 0.81 in IDC hearts. Furthermore, the allelic frequencies for the −9 and +9 alleles were 0.42 and 0.58 in normal hearts and 0.64 and 0.36 in IDC hearts, respectively. All analyzed CHD hearts were homozygous for the −9 allele. Thus, the expression of cardioprotective BK-2Rs in human hearts is increased with age in normal and IDC hearts and may be regulated on the transcriptional level. Moreover, comparison of normal subjects and patients with failing hearts revealed different allelic frequencies in each of two known BK-2R gene polymorphisms.