Nanoplaque Formation Research Articles

Abstract 363: The Brain - Pancreas Axis: An Alzheimer Nanoplaque Study

Background: The central-nervous regulation of blood glucose occurs purely neuroendocrine via the hypothalamus-hypophysis-adrenal cortex system. In this control loop, insulin is the adjustable and glucose the controlled variable. Methods: Ellipsometry, photometric methods and ELISAs were applied. Intracellular membrane potential and tension were recorded from human intracerebral arteries of consciousness areas. Results and discussion: Despite multiply feedback coupled and safeguarded control loops for maintenance of a constant blood glucose level, 4.7% of the world population suffered from diabetes mellitus (T2DM) in 1980 and already 8.5% in 2014. In parallel, the risk for cardiovascular (heart attack, stroke) and Alzheimer diseases (AD) has risen by a factor of four to eight. In ellipsometry measurements with polyanionic heparan sulfate proteoglycan (HS-PG), VLDLapoE4/E4 and IDLapoE4/E4, β-amyloid (1-42) and Ca 2+ -ions, we could show that non-enzymatic glycation of these substances leads to an Alzheimer nanoplaque formation increased by 20-30%. Since the viscoelastic polyelectrolyte HS-PG serves not only as lipoprotein receptor but also as blood flow sensor, we recorded with the same glycation a flow-dependent dilatation reduced by up to 80% in human brain arteries ex vivo (from neurosurgery) which express HS-PG in endothelial and smooth muscle cell membranes. The resulting O 2 -deficiency with certainty entails neuronal cell death. Postprandial, blood glucose levels can appear as were experimentally applied here. Conclusion: Also short-term distinctly increased blood glucose concentrations in apoE4 carriers can explain neuronal cell death like in AD.

Atherogenesis and plaque rupture, surface/interface-related phenomena

In atherogenesis, free oxygen radicals cause a lipid peroxidation of apoB100-containing lipoproteins in the blood, at the blood–endothelium-interface and in the subendothelial space. These lipoproteins easily aggregate, bind to their receptor heparan sulfate proteoglycan and calcify to arteriosclerotic nanoplaques (ternary complexes). Nanoplaque formation was measured by ellipsometry, both in vitro on an HS-PG coated hydrophobic silica surface and also in vivo on living human coronary endothelial cells, which had overgrown the silica surface. Reversely, we show with the same techniques that, in dependence on the degree of peroxidation and epitope in concern, oxLDL attacks its molecular receptor and thus can induce degradation of arteriosclerotic plaques and, in a combined action with inflammatory processes, even a plaque rupture.In a previous work, we had found PML-NB, fibrous cap (collagens, proteoglycans) and HSBGF binding sites (e.g., TGFβ1) up-regulated and NFκB down-regulated. With this background knowledge we created a molecular feedback control circuit model where PML-NB functions as regulation centre, fibrous cap as controlled variable, HSBGF binding sites as receptor and NFκB as effector. Since NFκB is inhibited by one reaction strand in this model and inhibits itself collagen and proteoglycan synthesis in the fibrous cap of the plaque, this double check (disinhibition) causes a stabilization of the fibrous cap through a specially strong collagen and proteoglycan production, which in addition is supported by circulating TGFβ. TGFβ furthers also calcification, so that fibrous cap tensile strength and resistance to shear stress are imparted. This way, a plaque rupture may possibly be averted.

Abstract 31: High-density Lipoprotein, a Feed-forward Forechecking Loop

Background: The protective effect of HDL is projected into the cholesterol back-transport of breakdown products of lipid metabolism to the liver. This is a feed-back circuit which controls cholesterol exit. Cholesterol entry could be safeguarded in a feed-forward forechecking loop at the proteoglycan (PG) receptor sites (syndecan, perlecan). HDL is the counter partner in the system of internalization and diffusion control for lipoprotein entry into the vascular wall. Based on stereochemical and chiral conformity with its PG receptor and the much higher negative charge density, HDL owns by far a higher affinity compared with LDL. Methods: Flow-dependent isometric tension, intracellularly recorded membrane potential and cAMP-cGMP were measured in segments of 25 coronaries from heart transplantations. Results and Discussion: Compared to Krebs solution, LDL (100 mg/dL) impaired flow-dilatation and caused a relative contraction by 18.5% (Table). In contrast, HDL (50 mg/dL) and HDL+LDL stimulated flow-dilatation by 31.1% and 41.4%, respectively (p < 0.96). Thus, the contractile effect of LDL was absent in the Krebs-HDL-LDL solution. The same effects were apparent in the membrane potential and cAMP-cGMP-concentrations of the VSM cells. These results are confirmed by ellipsometry measurements, where nanoplaque formation by LDL is suppressed by preincubation with HDL [Siegel, Malmsten, Ermilov: Adv Coll Interface Sci 205 (2014) 275-318]. Conclusion: LDL-cholesterol entry into the vascular wall is effectfully controlled by HDL in a feed-forward forechecking loop (HDL 4х higher affinity constant to the PG receptor). The interaction between both is dominated by competition.

Combined lowering of low grade systemic inflammation and insulin resistance in metabolic syndrome patients treated with Ginkgo biloba

In a clinical pilot study with eleven metabolic syndrome patients, a simultaneous decrease in hs-CRP from 8.85 ± 4.09 to 4.92 ± 2.51 mg/L (−44.4%) (p < 0.0436) and HOMA-IR from 3.07 ± 0.63 to 2.60 ± 0.51mU/L × mg/dL (−15.3%) (p < 0.0120) as well as a beneficial change of arteriosclerotic, inflammatory and oxidative stress biomarkers were detected after 2-month treatment with Ginkgo biloba. Furthermore, both IL-6 (−12.9%, p < 0.0407) and nanoplaque formation (−14.3%, p < 0.0077) were additionally reduced. According to a large clinical trial elucidating the importance of insulin resistance and low-grade systemic inflammation for cardiovascular disease and overall mortality risk, these data might indicate a CVD/total mortality risk reduction.

Abstract 408: The Importance of Vascular Risk Factors for Development of Dementia

Background: Since prevention and therapy of Alzheimer dementia turned out to be problematic, more attention should be paid to the vascular component of mixed pathologies, since this includes the possibility to decisively reduce the incidence of dementiae. ALP and GGT are indispensable constituents of brain microvessel endothelial cells, expressed and induced by IL-6. High values are entangled in affecting blood-brain barrier functionality. In a pilot study of 11 patients with metabolic syndrome, we examined whether these biomarkers can be beneficially influenced through an antioxidant phytochemical approach with Ginkgo biloba (EGb 761, 2 x 120 mg/d, 2 months). Methods: Photometric methods, ELISAs, EIAs and laser ellipsometry were applied. Results: Brain/liver biomarkers: ALP -14.8% (p &lt; 0.004), GGT -11.3% (&lt; 0.010). (Anti)oxidative biomarkers: SOD +17.7% (&lt; 0.009), GPx +11.6% (&lt; 0.001), oxLDL/LDL -21.0% (&lt; 0.002), 8-iso-PGF2α -39.8% (&lt; 0.003), MPO -29.6% (&lt; 0.014). Inflammatory biomarkers: IL-6 -12.9% (&lt; 0.041), Lp(a) -26.3% (&lt; 0.001), hs-CRP -39.3% (&lt; 0.005), WBC -6.3% (&lt; 0.023), MMP-9 -32.9% (&lt; 0.042). Vasodilator biomarkers: cAMP +43.5% (&lt; 0.001), cGMP +32.9% (&lt; 0.001), nanoplaque formation -14.3% (&lt; 0.008), nanoplaque size -23.4% (&lt; 0.0004). ALP and GGT were correlated to each other (r = 0.71; &lt; 0.0225) as well as to a decrease of oxidative, inflammatory and diabetic biomarkers (Table). Conclusion: Since under ginkgo, ALP and GGT were downregulated, SOD upregulated and plasma biomarkers beneficially altered, this phytochemical may be estimated as complementary drug with potentially preventive character for cardio-cerebro-vascular diseases including dementiae.

Abstract 033: Cardio-Cerebro-Vascular Diseases: Prevention in Metabolic Syndrome Patients

Background: ALP and GGT are indispensable constituents of brain microvessel endothelial cells, expressed and induced by IL-6. High values are entangled in affecting blood-brain barrier functionality. Since SOD bound to brain basement membrane and matrix heparan sulfate proteoglycan, is implicated in β-amyloid efflux, we examined in a pilot study of 11 patients with metabolic syndrome (MS) whether these biomarkers can be beneficially influenced through an antioxidant phytochemical approach with Ginkgo biloba (EGb 761, 2 × 120 mg/d). Also, MS is associated with an increased risk of developing diabetes and cardio-cerebro-vascular diseases (CCVD) (Figure). Methods: Photometric methods, ELISAs, EIAs and laser ellipsometry were applied. Results: A 2-month medication led to brain/liver biomarker changes: ALP -14.8% (p &lt; 0.004), GGT -11.3% (p &lt; 0.010). Antioxidative biomarker changes: SOD +17.7% (p &lt; 0.009), GPx +11.6% (p &lt;0.001), oxLDL/LDL -21.0% (p &lt; 0.002), 8- iso -PGF 2α -39.8% (p &lt; 0.003), MPO -29.6% (p &lt; 0.014). Antiinflammatory biomarker changes: IL-6 -12.9% (p &lt; 0.041), Lp(a) -26.3% (&lt; 0.001), hs-CRP -39.3% (&lt; 0.005), WBC -6.3% (&lt; 0.023), MMP-9 -32.9% (&lt; 0.042). Vasodilator biomarker changes: cAMP +43.5% (&lt; 0.001), cGMP +32.9% (&lt; 0.001), nanoplaque formation -14.3% (p &lt; 0.008), nanoplaque size -23.4% (p &lt; 0.0004). Conclusion: Since under ginkgo ALP and GGT were downregulated and SOD upregulated, and plasma biomarkers of oxidative stress, plaque stability and progression, inflammation, vasodilatory second messengers and lipid composition beneficially altered, this phytochemical may be estimated as complementary drug with potentially preventive character for CCVD.

The significance of lipid peroxidation in cardiovascular disease

BackgroundThe metabolic syndrome describes a cluster of cardiovascular risk factors that frequently appear together. Its diagnosis is generally based on several well-recognized indicators in clinical practice, such as abdominal obesity, elevated triglycerides, reduced high-density lipoprotein, raised blood pressure, and elevated fasting plasma glucose. Today, decisive importance must be attached to the metabolic syndrome since it leads to increased morbidity and mortality, and thus to a decreased life expectancy, and to higher direct and indirect healthcare costs. This is also due to the fact that its symptomatology irradiates on many organs of the body, which may thereby be damaged. MethodsIn the present clinical trial on 11 metabolic syndrome patients treated with Ginkgo biloba (EGb 761, 2×120mg/d) for two months, ellipsometry, fluorescence microscopy, photometric methods, ELISAs and EIAs were applied for biosensor profiling of metabolic syndrome risk, status and treatment outcome. ResultsA spectrum of more than 20 arteriosclerotic, cytokinic, inflammatory, lipidic, and oxidative stress biomarkers served for a detailed diagnosis and therapy monitoring. After medication, the ratio oxLDL/LDL was reduced by 21.0%, 8-iso-PGF2α 39.8%, MPO 29.6%, IL-6 12.9%, hs-CRP 39.3%, Lp(a) 26.3%, MMP-9 32.9%, insulin 9.4%, HOMA-IR 14.0%, ALP 14.8%, CREA 11.3%, URAC 10.6%, in vitro modeled nanoplaque formation 14.3% and size 23.4%, whereas SOD was augmented by 17.7%, GPx 11.6%, cAMP 43.5%, and cGMP 32.9%. Special focus was concentrated on the significance of lipid peroxidation for cardio-cerebro-vascular diseases. Through multiple correlations between the biomarkers and clinical parameters, their significance for and involvement in several clinical pictures could be elucidated. ConclusionThe present clinical observational study was helpful in unraveling this network of biomarker interactions and demonstrated its usefulness for theranostics. For personalized medicine, the selection of the biomarkers is of decisive importance. On the background of a growing obesity among children and adolescents with an increase in prevalence of the metabolic syndrome, diagnosing this syndrome in young subjects may be helpful in identifying a population of risk for increased subclinical arteriosclerosis.

Abstract 334: Complementary Atherosclerosis Prophylaxis in Patients After Coronary Bypass Surgery

Background and purpose: Atherosclerotic coronary arteries can be well-treated with innovative medicaments, through PCI or by aortocoronary bypasses. Some of the stents and bypasses close again in spite of blood-thinning. We examined in a clinical pilot study of 8 patients who had undergone a CABG operation whether atherosclerosis prophylaxis can be practised through an additional phytochemical approach with ginkgo (EGb 761, 2 × 120 mg/d). Furthermore, our trial aimed to inquire whether the bypasses remain open. Methods: As a biomarker for plaque development, atherosclerotic nanoplaque formation and size were monitored by laser ellipsometry (patent EP 0 946 876). Lipoproteins, cytosolic SOD, oxLDL, and [cAMP] and [cGMP] were determined by established methods. Results: After 2-month medication, the decrease in nanoplaque formation amounted to 11.9 ± 2.5% (p &lt; 0.01) and in nanoplaque size to 24.4 ± 8.1% (p &lt; 0.02), after 5 years 25.0 ± 3.7% (p &lt; 0.01) and 45.8 ± 9.5% (p &lt; 0.01), respectively. A multiple correlation between nanoplaque reduction and the risk factors oxLDL/LDL, Lp(a) and cGMP yielded an estimate of their relative contribution to nanoplaque diminution (34.7%, 43.3%, 22.0%). In the 5 year follow-up examination using high-resolution CT, all arterial and 86.7% of the venous bypasses were open, whereby the increase in blood nucleotides seemed to have played a prominent role. Conclusion: Ginkgo may be used as complementary drug with potentially preventive character after coronary-artery bypass graft.

Abstract 286: Phytochemical Treatment of Insulin Resistance in Metabolic Syndrome Patients

Background: Since HOMA-IR predicts coronary artery calcification incidence and progression, but not independently of metabolic syndrome (MS), we sought to determine whether insulin resistance (IR) could be lowered by a phytochemical approach. In a 2-month clinical pilot study with Ginkgo biloba (EGb 761, 2 × 120 mg/d) on 11 MS patients, a novel biomarker spectrum embracing parameters of plaque formation, stability and progression, oxidative stress, inflammation, lipid composition and second messengers, made point-of-care theranostics feasible. Methods: Laser ellipsometry, photometric methods, ELISAs and EIAs were applied. Results: Nanoplaque formation was reduced by 14.3% (p &lt; 0.01), nanoplaque size by 23.4% (p &lt; 0.0004), oxLDL/LDL 21.0% (p &lt; 0.002), 8- iso -PGF 2α 39.8% (p &lt; 0.003), MPO 29.6% (p &lt; 0.01), IL-6 12.9% (p &lt; 0.04), hs-CRP 39.3% (p &lt; 0.005), Lp(a) 26.3% (p &lt; 0.001), MMP-9 32.9% (p &lt; 0.04), whereas SOD was augmented 17.7% (p &lt; 0.01), GPx 11.6% (p &lt; 0.001), cAMP 43.5% (p &lt; 0.001), and cGMP 32.9% (p &lt; 0.001). Since none of the patients had a diabetes, we evaluated IR. Fasting morning glucose was reduced by 4.0% from 94.4 to 90.2 mg/dL (p &lt; 0.03), insulin 8.6% from 12.7 to 11.5 mU/L (p &lt; 0.04), and HOMA-IR 12.3% from 3.07 to 2.64 mU/L×mg/dL (p &lt; 0.02). Because ginkgo is not an antidiabeticum, we looked out for a mechanistic explanation. Insulin (1), glucose (2) and HOMA-IR (3) were correlated to IL-6 (4), hs-CRP (5), TNFα (6) and TGFβ 1 (7), which changes in cytokine pattern could unravel lowering of the former quantities: 1 vs 4 (r = 0.73, p &lt; 0.06); 2 vs 4 (r = 0.80, p &lt; 0.03); 3 vs 4 (r = 0.71, p &lt; 0.07); 1 vs 5 (r = 0.64, p &lt; 0.03); 2 vs 5 (r = 0.75, p &lt; 0.02); 3 vs 5 (r = 0.68, p &lt; 0.14); 1 vs 6 (r = -0.68, p &lt; 0.05); 2 vs 6 (r = -0.73, p &lt; 0.04); 3 vs 6 (r = -0.67, p &lt; 0.05); 1 vs 7 (r = -0.75, p &lt; 0.09); 2 vs 7 (r = -0.70, p &lt; 0.02) and 3 vs 7 (r = -0.89, p &lt; 0.01). Conclusion: Ginkgo had beneficial effects on a multitude of arteriosclerotic and diabetic biomarkers. IR was diminished by 12.3% to an HOMA-IR markedly below 3 on average, a clinically tolerable level. Further, this decrease can explain the reduction in Ca 2+ -dependent nanoplaque formation and size. Thus, ginkgo may be used as complementary drug in the treatment of MS, arteriosclerotic and diabetic patients.

Abstract P356: In Vitro Effect of Blackcurrant Anthocyanins on Flow-Dependent Dilatation of Human Intracerebral Arteries and Alzheimer Nanoplaque Formation

Background: Vascular wall amyloidosis, one of the important cell and tissue changes in dementiae of the Alzheimer type, is characterized by massive deposition of β-amyloid in the walls of leptomeningeal and cortical arteries and arterioles, as well as in vessels in the brainstem and cerebellum. This deposition depends critically on both lipoprotein isoform and cholesterol, which has prompted research on the link between coronary disease and Alzheimer's disease. High cholesterol diets were found to induce accumulation of β-amyloid in the brain. Anthocyanins from blackcurrant extract (BCE) may have beneficial effects on Alzheimer dementiae via other target points than cholesterol lowering. Materials and Methods: Flow-dependent isometric tension was measured in segments of isolated human intracerebral arteries from consciousness areas, coming from brain surgery. The blood vessel segments were stretched by 1.5 g pretension. The flow of the blood substitute solution (Krebs) was varied in the steps 3, 5, 20, 40, and 100 mL/min. Krebs solution without and with addition of 0.01% BCE (BC ACL-1.5, BerryPharma AG, Leichlingen, Germany) was used as superfusate. Nanotechnologic biosensor ellipsometry, photometric methods, ELISAs and EIAs were applied. VLDLapoE4/E4 (10 mg/dL) from genotypized patients and human β-amyloid peptide (1-42) (Aβ) were used in the ellipsometric measurements. Results: In the controls (n = 14), the smooth muscle cells of the brain arteries relaxed from 1.416 ± 0.009 g to 1.011 ± 0.033 g (p &lt; 0.001) corresponding to 28.6% of their initial tone. Under blackcurrant liquid extract (n = 5), the decrease in wall tension was much more distinct. Vascular tone decreased from 1.454 ± 0.010 g (flow 3 mL/min) to 0.867 ± 0.052 g (flow 100 mL/min) (p &lt; 0.001; against the control p &lt; 0.0354). This is equivalent to a 40.4% reduction in tension, a 45.2% increase in flow-dependent relaxation, and an estimated 50.7% rise in blood perfusion under blackcurrant. BCE (0.001%) is strongly bound to membrane integral proteoheparan sulphate (HS-PG), the natural flow sensor and peripheral lipoprotein receptor, as measured by ellipsometric techniques. Addition of VLDLapoE4/E4, Aβ, and Ca 2+ ions (2.52 to 17.64 mmol/L) to HS-PG adsorbed to a silica surface led to quaternary Alzheimer plaque formation in control experiments. Preincubation of HS-PG with BCE reduced VLDL docking by 5.9%, ternary plaque formation with Aβ by 11.6%, and quaternary calcified Alzheimer nanoplaque formation by 17.3%. Conclusion: The flow experiments impressively show that BCE clearly improves endothelial function by an additional NO release. This vasodilatation together with the reduction of Alzheimer nanoplaque neoformation, detected here for the first time as a novel pleiotropic action of BCE, may have a beneficial effect on the cognitive functions in dementiae of the Alzheimer type, in the prevention of TIA and stroke.

The role of the proteoglycan receptor in lipoprotein binding

Heparan sulfate proteoglycan can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution representing one receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. A second lipoprotein binding site has been localized to four copies of LDL receptor repeats of core domain II. The binding process was studied by ellipsometric techniques showing that HDL has a high binding affinity to the receptor and a protective effect on interfacial heparan sulfate proteoglycan layers with respect to LDL and Ca2+ complexation. LDL was found to deposit strongly at the proteoheparan sulfate, particularly in the presence of Ca2+, thus creating the complex formation ‘proteoglycan—low density lipoprotein—calcium’. This ternary complex build-up may be interpreted as arteriosclerotic nanoplaque formation on the molecular level responsible for the arteriosclerotic primary lesion. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL was able to both reduce the ternary complex deposition and to disintegrate HS-PG/LDL/Ca2+ aggregates. Therefore, HDL attached to its proteoglycan receptor sites is thought to raise a multidomain barrier, selection and control motif for transmembrane and paracellular lipoprotein uptake into the arterial wall. Although much remains unclear regarding the mechanism of lipoprotein depositions at proteoglycan-coated surfaces, it seems clear that the use of such systems offers possibilities for investigating lipoprotein deposition at a ‘nanoscopic’ level under close to physiological conditions. In particular, Ca2+-promoted LDL deposition and the protective effect of HDL, even at high Ca2+ and LDL concentrations, agree well with previous clinical observations regarding risk and beneficial factors for early stages of atherosclerosis.

The importance of scavenging reactive oxygen species in anti‐aging medicine

AbstractIn a pilot study, we had reported on the beneficial effects of Ginkgo biloba (EGb 761) on arteriosclerotic nanoplaque formation and size in cardiovascular high‐risk patients who had undergone an aortocoronary bypass operation. Briefly, nanoplaque formation and size, the ratio oxLDL/LDL, and the highly atherothrombotic lipoprotein(a) concentration were substantially reduced, while superoxide dismutase activity and the blood concentration of the vasodilating substances cAMP and cGMP were upregulated. Since the arteriosclerosis prophylactic and well‐aging promotive impact of Ginkgo extract has been proven in this pilot study, we wanted to confirm these beneficial effects through a second observational clinical trial. The measurable variables formerly used were additionally supplemented by a wide, novel biomarker spectrum, through which the latest parameters and markers of plaque stability and progression, oxidative stress, and inflammation were available. In eleven patients with metabolic syndrome in the initial stage, the reduction of arteriosclerotic nanoplaque formation amounted to 14.3±2.9% (p&lt;0.0077) and of nanoplaque size to 23.4±3.7% (p&lt;0.0004), respectively, after 2‐months of treatment with Ginkgo biloba extract. Additionally, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were upregulated by 19.6±10.0% (p&lt;0.0785) and 11.6±2.3% (p&lt;0.001), respectively, the quotient oxLDL/LDL lowered by 21.0±4.3% (p&lt;0.002), and lipoprotein(a) concentration decreased by 26.3±4.8% (p&lt;0.001) in the patients' blood. The concentration of cAMP and cGMP was augmented by 43.5±12.0% (p&lt;0.001) and 32.9±10.4% (p&lt;0.001), respectively. Surprisingly, we found a lowering of the serum Ca2+ concentration by 5.4±1.6% (p&lt;0.0076) from 2.37±0.03 to 2.24±0.04 mmol/L (p&lt;0.0069). Apart from an additional vasodilatory effect, the lowered extracellular Ca2+ concentration affects nanoplaque formation restrictively, since this is a Ca2+ driven process. Furthermore, we could show a favourable development of the biomarkers 8‐iso‐PGF2α, oxLDL/LDL, SOD, GPx (oxidative stress), hs‐CRP, MPO, TNFα, TGFβ1 (inflammatory status) and MMP‐9 (plaque stability). The markers selected here are suited to provide a comprehensive risk profile for the prevention of arteriosclerosis. Finally, a multiple regression analysis reveals a basis for a mechanistic explanation of nanoplaque reduction under Ginkgo treatment. The arteriosclerosis inhibiting effect is due to an attenuation of the risk factors oxLDL/LDL, Lp(a), and [Ca2+]o as well as to a significant increase in the vasodilator cAMP and cGMP concentration. Thus, Ginkgo with its pleiotropic effects should be assigned a fixed rank among the anti‐aging medical therapeutics as a prophylactic measure, especially in patients with early‐stage metabolic syndrome.

Reduction of Arteriosclerotic Nanoplaque Formation and Size by n-3 Fatty Acids in Patients after Valvular Defect Operation

Background/Methods: Coating a silica surface with the isolated lipoprotein receptor heparan sulfate proteoglycan (HS-PG) from arterial endothelium and vascular matrices, we could observe the very earliest stages of arteriosclerotic plaque development by ellipsometric techniques in vitro (patent EP 0 946 876). This so-called nanoplaque formation is represented by the ternary aggregational complex of the HS-PG receptor, lipoprotein particles and calcium ions. The model was validated in several clinical studies on statins in cardiovascular high-risk patients applying their native blood lipoprotein fractions. Results: In 7 patients who had undergone a valvular defect operation, the reduction of arteriosclerotic nanoplaque formation in normal Krebs solution amounted to 6.1 ± 2.3% (p < 0.0156) and of nanoplaque size to 37.5 ± 13.2% (p < 0.0312), respectively, after a 3-month therapy with n-3 fatty acids (3 ··3 g daily, Ameu® 500 mg). Additionally, the quotient oxLDL/LDL was lowered by 6.8 ± 2.1% (p < 0.0166), the MDA concentration remained unchanged and the lipoprotein(a) concentration decreased by 15.8 ± 5.6% (p < 0.0469) in the patients’ blood. The concentration of the nanoplaque promoting particles VLDL and total triglycerides was diminished by 34.1 ± 11.6% (p < 0.0469) and 26.7 ± 10.8% (p < 0.0156), respectively. Furthermore, the ratio of the strongly atherogenic small dense to the total LDL cholesterol (LDL5+LDL6)/LDLtot decreased by 9.9 ± 3.0% (p < 0.0174). Conclusions: A combinatorial regression analysis revealed a basis for a mechanistic explanation of nanoplaque reduction under n-3 fatty acid treatment. This effect was possibly due to the beneficial changes in lipid concentrations and an attenuation of the risk factors oxLDL/LDL and (LDL5+LDL6)/LDLtot.

Nanotechnologic biosensor ellipsometry and biomarker pattern analysis in the evaluation of atherosclerotic risk profile

A proteoheparan sulfate coated, hydrophobic silica surface serves as lipoprotein receptor at which the Ca 2+-driven arteriosclerotic nanoplaque formation can be pursued by laser-based ellipsometry. Any lipoprotein from human blood can be very sensitively tested for its atherogenic properties. From the same blood sample, it is possible to determine the concentration and activity of a series of interacting biomarker molecules which, through a pattern analysis, allow to assess the state of health with respect to cardiovascular diseases. These two interlinked and complementary biosensors make a prospective cardio-cerebro-vascular risk stratification feasible, especially the sequelae of an underlying arteriosclerotic disease. Based on these diagnostic tools, an optimized therapy decision for the patient can be taken and the necessary preventive measures for the still healthy person.

Reduction of atherosclerotic nanoplaque formation and size by Ginkgo biloba (EGb 761) in cardiovascular high-risk patients

Coating a silica surface with the isolated lipoprotein receptor proteoheparan sulfate (HS-PG) from arterial endothelium and vascular matrices and adding both the atherogenic VLDL/IDL/LDL lipid fraction in its native composition and Ca 2+ ions, we could observe in vitro the earliest stages of atherosclerotic plaque development by ellipsometric techniques (patent EP 0 946 876). This so-called nanoplaque formation is represented by the ternary aggregational complex of the HS-PG receptor, lipoprotein particles and calcium ions. The model was validated in several clinical studies on statins in cardiovascular high-risk patients. In eight patients who had undergone an aortocoronary bypass operation, the reduction of atherosclerotic nanoplaque formation amounted to 11.9 ± 2.5% ( p < 0.0078) and of nanoplaque size to 24.4 ± 8.1% ( p < 0.0234), respectively, after a 2-month therapy with Ginkgo biloba extract (2× 120 mg daily, EGb 761). Additionally, superoxide dismutase (SOD) activity was upregulated by 15.7 ± 7.0% ( p < 0.0391), the quotient oxLDL/LDL lowered by 17.0 ± 5.5% ( p < 0.0234) and lipoprotein(a) concentration decreased by 23.4 ± 7.9% ( p < 0.0234) in the patients’ blood. The concentration of the vasodilating substances cAMP and cGMP was augmented by 37.5 ± 9.1% ( p < 0.0078) and 27.7 ± 8.3% ( p < 0.0156), respectively. A multiple regression analysis between the patients’ VLDL/IDL/LDL lipoprotein fraction applied in the ellipsometry measurements as well as the further risk factors oxLDL/LDL and Lp(a) on the one hand and changes in nanoplaque formation on the other hand reveals a basis for a mechanistic explanation of nanoplaque reduction under ginkgo treatment. The atherosclerosis inhibiting effect is possibly due to an upregulation in the body's own radical scavenging enzymes and an attenuation of the risk factors oxLDL/LDL and Lp(a).

Towards biosensing of arteriosclerotic nanoplaque formation using femtosecond spectroscopy

The ultrafast dynamics of proteoheparan sulfate (HS-PG) in Krebs blood substitute solution was measured using femtosecond transient absorption spectroscopy after UV excitation. Interacting with blood lipoproteins and Ca 2+ ions, the proteoglycan HS-PG is the key component of the so-called nanoplaque, the earliest stage in atherogenesis. Since tryptophan (Trp) residues are the main optically active parts of HS-PG, analogous measurements were performed on bare Trp in Krebs solution. The comparison reveals distinct differences to main characteristics of the HS-PG broadband absorption spectra. Analyzing the Trp spectra, we show that the results from transient absorption spectroscopy resemble the time constants of the chromophore ultrafast solvation dynamics that have been found by another group using fluorescence up-conversion techniques. Yet, the broadband transient absorption provides more details about the molecular dynamics, including stimulated emission, excited state absorption and resonant energy transfer. Furthermore, the absorption long time dynamics upon adding Ca 2+ to the HS-PG probe were investigated by transient absorption spectroscopy and by surface force and ellipsometry investigations. Notably, a Ca 2+-induced conformational change responsible for arteriosclerotic nanoplaque formation was detected. Slight differences, which are only visible as broad spectral features in the sub-picosecond time scale, provide a first insight into the molecular formation of nanoplaques in blood vessels, which may yield a better understanding of the genesis of arteriosclerosis.

Ginkgo biloba (EGb 761) in arteriosclerosis prophylaxis

The prevention or deceleration of atherogenesis is one of the most significant anti-aging objectives since this is a matter of avoidance of myocardial infarction and stroke. To approach this prophylactic aim, phytochemical nutrition counteracting peroxidation of blood lipids based on their scavenger qualities for reactive oxygen species (ROS) can possibly serve. For example, oxidized LDL particles are highly atherogenic. Against this background, we investigated in a pilot study the effect of Ginkgo biloba (EGb 761: Rökan novo), the free oxygen radical scavenging properties of which are well-documented, on the atherosclerotic nanoplaque formation in cardiovascular high-risk patients. In eight patients who had undergone an aortocoronary bypass operation, the reduction of atherosclerotic nanoplaque formation amounted to 11.9 +/- 2.5% (p < 0.0078) and of nanoplaque size to 24.4 +/- 8.1% (p < 0.0234), respectively, after a 2-month therapy with Ginkgo biloba extract (EGb 761, 2 x 120 mg daily, Rökan novo, Spitzner Arzneimittel, Ettlingen, Germany). Additionally, superoxide dismutase (SOD) activity was upregulated by 15.7 +/- 7.0% (p < 0.0391), the quotient oxLDL/LDL lowered by 17.0 +/- 5.5% (p < 0.0234) and lipoprotein(a) concentration decreased by 23.4 +/- 7.9% (p < 0.0234) in the patients' blood after the 2-month medication regimen. The concentration of the vasodilating substances cAMP and cGMP was augmented by 37.5 +/- 9.1% (p < 0.0078) and 27.7 +/- 8.3% (p < 0.0156), respectively. A multimodal regression analysis reveals a basis for a mechanistic explanation of nanoplaque reduction under ginkgo treatment. The atherosclerosis inhibiting effect is due to an upregulation in the body's own radical scavenging enzymes and an attenuation of the risk factors oxLDL/LDL and Lp(a). Furthermore, the significant increase in the vasodilator cAMP and cGMP concentration powerfully supports the maintenance of an open bypass.

The effect of Ginkgo biloba (EGb 761) on arteriosclerotic nanoplaque formation and size in a long-term clinical trial

Abstract Utilizing the isolated lipoprotein receptor syndecan (heparan/chondroitin sulfate proteoglycan, HS/CS-PG) from arterial endothelium and smooth muscle cell membranes and coating therewith a silica surface, we were able to observe the very earliest stages of arteriosclerotic plaque development, the so-called nanoplaque build-up, by ellipsometric techniques (patent EP 0 946 876). The arteriosclerotic nanoplaque is represented by the ternary aggregational complex of the HS-PG receptor, lipoprotein particles and calcium ions. The model was validated in several clinical studies [1,2] on cardiovascular high-risk patients applying their blood lipoprotein fractions and, among others, proved the inhibiting effect of Ginkgo biloba on nanoplaque formation. In eight high-risk patients who had undergone an aortocoronary bypass operation, the reduction of arteriosclerotic nanoplaque formation amounted to 11.9 ± 2.5% ( p ≤ 0.0078, non-parametric Wilcoxon-test for paired samples; median 10.6%) and of nanoplaque size to 24.4 ± 8.1% ( p ≤ 0.0234, non-parametric Wilcoxon-test for paired samples; median 20.4%), respectively, in normal blood substitute solution with 2.5 mmol/l [Ca 2+ ] after a 2 month therapy with 2 × 120 mg Ginkgo biloba extract (EGb 761, Rokan® novo). Additionally, we could directly demonstrate and confirm the antioxidative capacity of ginkgo and its oxygen free radical scavenging effect by disclosing an upregulation of superoxide dismutase (SOD) activity of 15.7 ± 7.0% ( p ≤ 0.0391, non-parametric Wilcoxon-test for paired samples; median 16.2%) and a lowering of the quotient oxLDL/LDL by 17.0 ± 5.5% ( p ≤ 0.0234, non-parametric Wilcoxon-test for paired samples; median 16.9%) after the 2 month medication regimen. Furthermore, we measured a significant decrease in lipoprotein(a) concentration falling from 52.4 ± 8.2 to 42.0 ± 9.9 mg/dl ( p ≤ 0.0359, paired Student t-test). Altogether, these beneficial effects of Ginkgo biloba might have partially repaired endothelial dysfunction being responsible for the very earliest stages in arteriosclerosis and could present a basis for a mechanistic explanation of nanoplaque reduction under Ginkgo treatment. This further pleiotropic effect of Ginkgo illuminated for the first time in this biosensor-biomembrane approach has immediate relevance for the formation of arteriosclerotic plaques as well as for its prophylactic and therapeutic potential in patients.

Hemmung der arteriosklerotischen Plaqueentstehung durch Knoblauch

In an in vitro biosensor model (PCT/EP 97/05212), the interplay between different lipoproteins in arteriosclerotic nanoplaque formation, as well as aqueous garlic extract (0.2-5.0 g/l from LI 111 powder) as a possible candidate drug against arterio/atherosclerosis were tested within the frame of a high throughput screening. The processes described below were studied by ellipsometric techniques quantifying the adsorbed amount (nanoplaque formation) and layer thickness (nanoplaque size). A thorough description of the experimental setup has been given previously. Proteoheparan sulfate (HS-PG) adsorption to hydrophobic silica was monoexponential and after approximately 30 min constant. The LDL plasma fraction (100 mg/dl) from a healthy probationer showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca2+ concentration, with a strong increase at higher Ca2+ concentrations. Aqueous garlic extract (GE), preferably in a concentration of 1 g/l, applied acutely in the experiment, markedly slowed down this process of ternary aggregational nanoplaque complexation at all Ca2+ concentrations used. In a normal blood Ca2+ concentration of 2.52 mmol/l, the garlic induced reduction of nanoplaque formation and molecular size amounted to 14.8 % and 3.9%, respectively, as compared to the controls. Furthermore, after ternary complex build-up, GE similar to HDL, was able to reduce nanoplaque formation and size. The incubation time for HDL and garlic was only 30 min each in these experiments. Nevertheless, after this short time the deposition of the ternary complex decreased by 6.2% resp. 16.5%, i.e. the complex aggregates were basically resolvable. These experiments clearly proved that garlic extract strongly inhibits Ca2+ binding to HS-PG. In consequence, the formation of the ternary HS-PG/LDL/Ca2+ complex, initially responsible for the 'nanoplaque' composition and ultimately for the arteriosclerotic plaque generation, is decisively blunted.

The effect of garlic on arteriosclerotic nanoplaque formation and size.

In an in vitro biosensor model (PCT/EP 97/05212), the interplay between different lipoproteins in arteriosclerotic nanoplaque formation, as well as aqueous garlic extract (0.2-5.0 g/l from LI 111 powder) as a possible candidate drug against arterio/atherosclerosis were tested within the frame of a high throughput screening. The processes described below were studied by ellipsometric techniques quantifying the adsorbed amount (nanoplaque formation) and layer thickness (nanoplaque size). A thorough description of the experimental setup has been given previously. Proteoheparan sulfate (HS-PG) adsorption to hydrophobic silica was monoexponential and after approximately 30 min constant. The addition of 2.52 mmol/l Ca2+ led to a further increase in HS-PG adsorption because Ca2+ was bound to the polyanionic glycosaminoglycan (GAG) chains thus screening their negative fixed charges and turning the whole molecule more hydrophobic. Incubation with 0.2 g/l aqueous garlic extract (GE) for 30 min did not change the adsorption of HS-PG. However, the following addition of Ca2+ ions reduced the increase in adsorption by 50.8% within 40 min. The adsorption of a second Ca2+ step to 10.08 mmol/l was reduced by even 82.1% within the next 40 min. Having detected this inhibition of receptor calcification, it could be expected that the build-up of the ternary nanoplaque complex is also affected by garlic. The LDL plasma fraction (100 mg/dl) from a healthy probationer showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca2+ concentration, with a strong increase at higher Ca2+ concentrations. GE, preferably in a concentration of 1 g/l, applied acutely in the experiment, markedly slowed down this process of ternary aggregational nanoplaque complexation at all Ca2+ concentrations used. In a normal blood Ca2+ concentration of 2.52 mmol/l, the garlic induced reduction of nanoplaque formation and molecular size amounted to 14.8% and 3.9%, respectively, as compared to the controls. Furthermore, after ternary complex build-up, GE similar to HDL, was able to reduce nanoplaque formation and size. The incubation time for HDL and garlic was only 30 min each in these experiments. Nevertheless, after this short time the deposition of the ternary complex decreased by 6.2% resp. 16.5%, i.e. the complex aggregates were basically resolvable. These experiments clearly proved that garlic extract strongly inhibits Ca2+ binding to HS-PG. In consequence, the formation of the ternary HS-PG/LDL/Ca2+ complex, initially responsible for the 'nanoplaque' composition and ultimately for the arteriosclerotic plaque generation, is decisively blunted.