The effect of an HMG-CoA reductase inhibitor on arteriosclerotic nanoplaque formation and size in a biosensor model.

Proteoheparan sulfate 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, thereby representing a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques. Low-density lipoprotein (LDL) was found to deposit strongly at the proteoheparan sulfate-coated surface, particularly in the presence of Ca(2+), apparently through complex formation 'proteoglycan-LDL-calcium'. This ternary complex build-up may be interpreted as arteriosclerotic nanoplaque formation on the molecular level responsible for the arteriosclerotic primary lesion. HDL bound to heparan sulfate proteoglycan protected against LDL deposition and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL was able to decelerate the ternary complex deposition and to disrupt newly formed nanoplaques. 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. The molecular arteriosclerosis model was tested on its reliability in a biosensor application in order to unveil possible acute pleiotropic effects of the lipid lowering drug fluvastatin. The very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL)/LDL and VLDL/IDL/LDL/HDL plasma fractions from a high-risk patient with dyslipoproteinemia and type 2 diabetes mellitus showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca(2+) concentration, with a strong increase at higher Ca(2+) concentrations. Nanoplaque formation and size of the HDL-containing lipid fraction remained well below that of the LDL-containing lipid fraction. Fluvastatin, whether applied acutely to the patient (one single 80 mg slow release matrix tablet) or in a 2-months medication regimen, markedly slowed down this process of ternary aggregational nanoplaque build-up and substantially inhibited nanoplaque size development at all Ca(2+) concentrations used. The acute action resulted without any significant change in lipid concentrations of the patient. Furthermore, after nanoplaque generation, fluvastatin, similar to HDL, was able to reduce nanoplaque formation and size. These immediate effects of fluvastatin have to be taken into consideration while interpreting the clinical outcome of long-term studies.

Biosensing of arteriosclerotic nanoplaque formation and interaction with an HMG-CoA reductase inhibitor.

Proteoheparan sulphate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. As a result of electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution, thereby representing one receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques suggesting that high-density lipoprotein (HDL) has a high binding affinity and a protective effect on interfacial heparan sulphate proteoglycan layers with respect to low-density lipoprotein (LDL) and Ca2+ complexation. Low-density lipoprotein was found to deposit strongly at the proteoheparan sulphate-coated surface, particularly in the presence of Ca2+, apparently through complex formation 'proteoglycan-LDL-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 sulphate proteoglycan protected against LDL deposition and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL was able to decelerate the ternary complex deposition. 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. Considering this, the system was tested on its reliability in a biosensor application in order to unveil possible acute pleiotropic effects of the lipid lowering drug fluvastatin. The very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL)/LDL plasma fraction from a high risk patient with dyslipoproteinaemia and type 2 diabetes mellitus showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca2+ concentration, with a strong increase at higher Ca2+ concentrations. Fluvastatin, whether applied to the patient (one single 80 mg slow release matrix tablet) or acutely in the experiment (2.2 micromol L-1), markedly slowed down this process of ternary aggregational nanoplaque complexation at all Ca2+ concentrations used. This action resulted without any significant change in lipid concentrations of the patient. Furthermore, after ternary complex build-up, fluvastatin, similar to HDL, was able to reduce nanoplaque adsorption and size. These immediate effects of fluvastatin have to be taken into consideration while interpreting the clinical outcome of long-term studies.

A receptor-based biosensor for lipoprotein docking at the endothelial surface and vascular matrix.

Proteoheparan sulfate 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 a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. 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 Ca(2+) complexation. LDL was found to deposit strongly at the proteoheparan sulfate, particularly in the presence of Ca(2+), 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 and aqueous garlic extract were able to reduce the ternary complex deposition and to disintegrate HS-PG/LDL/Ca(2+) aggregates. 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, Ca(2+)-promoted LDL deposition and the protective effect of HDL, even at high Ca(2+) and LDL concentrations, agree well with previous clinical observations regarding risk and beneficial factors for early stages of atherosclerosis. Therefore, we believe that the system can be of some use in investigations, e.g. of the interplay between different lipoproteins in arteriosclerotic plaque formation, as well as in high throughput screening of candidate drugs to atherosclerosis in a biosensor application.

Physicochemical binding properties of the proteoglycan receptor for serum lipoproteins.

Proteoheparan sulfate 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 polysugar side chains are stretched out into the blood substitute solution representing a co-receptor for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that oxLDL had a deleterious effect on heparan sulfate proteoglycan binding and conformation. Ca2+ binding to and storage on the proteoheparan sulfate/LDL compound formed a 'heterotrimeric' HS-PG/LDL/Ca2+ complex of high stability, aggregability and deposit coating. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan/lipoprotein complex.

The antiatherosclerotic effect of Allium sativum.

In a randomized, double-blind, placebo-controlled clinical trial, the plaque volumes in both carotid and femoral arteries of 152 probationers were determined by B-mode ultrasound. Continuous intake of high-dose garlic powder dragees reduced significantly the increase in arteriosclerotic plaque volume by 5-18% or even effected a slight regression within the observational period of 48 months. Also the age-dependent representation of the plaque volume shows an increase between 50 and 80 years that is diminished under garlic treatment by 6-13% related to 4 years. It seems even more important that with garlic application the plaque volume in the whole collective remained practically constant within the age-span of 50-80 years. These results substantiated that not only a preventive but possibly also a curative role in arteriosclerosis therapy (plaque regression) may be ascribed to garlic remedies.

Tumor cell locomotion and metastatic spread.

The cytoskeletal filament proteins alpha-actinin, filamin, desmin, and filamin-desmin aggregates were adsorbed to a hydrophobic silica surface. The adsorbed amount as measured by ellipsometric methods after rinsing and equilibration was 2.7 mg/m2 for alpha-actinin and 0.4 mg/m2 for filamin plus desmin, respectively. Adsorbed layer thicknesses in physiological salt solution were about 107 nm, 89 nm, 108 nm and 93 nm for alpha-actinin, filamin, desmin, and cross-linked filamin-desmin, respectively. Ca2+ ions in a concentration of 10(-4), 10(-3), and 2.52 mmol/l had no effect on the adsorbed amount, refractive index, and adsorbed layer thickness of the individual intermediate filament proteins. Cross-linked filamin-desmin, however, reacted markedly upon the addition of these Ca2+ concentrations with a change in refractive index and adsorbed layer thickness. The layer formed by the filamin-desmin complex contracted by 2-3, 6-7, and 6-7 nm, respectively. The maximum shortening occurred at 1 pmol/l Ca2+. The Ca(2+)-dependent adsorbed layer changes of cross-linked filamin-desmin supports the contractile mechanisms in muscular tissues and forms the basis for migration and motility in nonmuscular cells. These motional events are crucially involved in peripheral organ perfusion, inflammation, and tumor invasion and metastasis.

Vascular smooth muscle, a multiply feedback-coupled system of high versatility, modulation and cell-signaling variability.

Under normal conditions, the various vascular regulatory effector influences are interwoven in a dynamic, and not a static, circulatory system. The reaction of a smooth muscle cell is thus reflected only incompletely by the stationary activation curve 'developed tension versus membrane potential'. The missing time domain in this relationship is a reflection of our as yet limited understanding of the system's behavior in space and time. It should be emphasized that the rhythmogenic properties of vascular smooth muscle are closely coupled to a functioning circulation. The electrical and mechanical oscillations, which can be traced back to rhythmic activity of the active, electrogenic Na+/K+ pump, could originate in the allosteric qualities of the enzyme phosphofructokinase (PFK). Thus, PFK represents a rhythmogenic enzyme which may serve as an example of the connection between the biological properties on a molecular level and the spatiotemporal system's behavior. The cardiovascular system and its rhythmicity may be dominated by only a few control points, one of which is distinguished by the viscoelastic properties of a blood flow sensor macromolecule. Therefore, the three prominent control points - PFK, (Na+ + K+)-ATPase and flow sensor conformation - acting as negatively feedback-coupled, nonlinear synergetic order parameters, are sufficient to initiate the periodic events in the cardiovascular system and to provide a plausible explanation for their causal origin.

Blood-flow sensing by anionic biopolymers.

Using 23Na-NMR techniques we could show that the polyanion proteoheparan sulfate integrated into the membrane of endothelial cells may serve as "flow sensor'. Based on its viscoelastic properties, heparan sulfate proteoglycan is present as a random coil under "no flow' conditions, whereby most of its polyanionic sites undergo intramolecular hydrogen bonding. With increasing flow the macromolecule becomes unfolded into a filamentous structure. Additional anionic binding sites to which Na+ ions from the blood bind are released by this shear stress-dependent conformational change. The Na+ binding triggers the signal transduction chain for a vasodilatory vessel reaction. Decrease in flow effects, for reasons of the intramolecular elastic recoil forces of the macromolecules, an entropic coiling, the release of Na+ ions and thus an interruption of the signal chain. Proteoheparan sulfate adsorbed onto a hydrophobic surface in physiological Krebs solution at pH 7.3 demonstrated clearly its characteristic as a Na+ sensor. While Ca2+ ions modulated the adsorption (promotion with increasing Ca2+ concentrations) by changing the conformation of the sensor molecule, the adsorbed amount was determined preferably by the Na+ concentration. K+ and Mg2+ ions showed slightly desorbing properties with increasing concentrations. Thus, it may be concluded that Na+ ions play the role as "first messenger' in flow-dependent vasodilation.

Integrative neurovegetative and motor control: phenomena and theory.

The general problem of specificity and the existence of separate central control systems for different peripheral systems is handled and exemplified by the control of circulation, respiration and motor innervation. The considerations are based on several earlier experimental studies of spontaneous coordinations of rhythmic activities in anaesthetized dogs and conscious men and microelectrode recordings and local cooling experiments in the brain stem of anaesthetized dogs and cats. Analysis of findings and logical deductions result in the statement that central control systems must be partly identical and that the usual criteria of labelling central neurones according to their coordination to specific control systems are necessary but not sufficient. Instead the picture of a multifunctional common network emerges forming the substrate of the observed common central rhythmicity. In this network specificity is a quantitative property of neurones. A simple model is outlined illustrating that slight inhomogeneity leads to graded input-output specificity in the network which changes with changing conditions. The degree of neuronal specificity increases in the direction of the outputs. Applications of the network model to concrete observations in animal experiments are discussed.

Neurophysiological background of central neural cardiovascular-respiratory coordination: basic remarks and experimental approach.

This paper is comprised of a review of past contributions and their theoretical consequences and a presentation of new results in studies of the origin of sympathetic rhythms and tone. Two basic mechanisms are involved: a primary intracentral coupling with the main cardiovascular-respiratory rhythm (MCRR) generator and a secondary reflex coupling. It was found that the activity and rhythms of certain sympathetic efferents, such as the cervical sympathetic, are closely related to the MCRR. Other efferents, such as the renal sympathetics, are loosely linked and follow drives from other circuits in the genesis of their rhythms and tone. New experimental evidence is given that rhythmic and tonic activity in sympathetic nerves originates from several sources. Hence central respiratory-autonomic systems interaction is not explained by simplistic concepts.

Mechanisms of central transmission of respiratory reflexes.

Several types of respiratory reflex actions can be discerned according to the reactions of typical respiratory neurons in the efferent part of the central rhythmogenic structure. Whereas respiration runs closely parallel with inspiratory neuron activity the behaviour of expiratory neurons cannot be derived from the resulting reflex changes of respiration. So expiratory apnoea can be combined with continuous activity or with inactivation of expiratory neurons. Extracellular records from a closed uniformly reacting population of expiratory neurons and from neighbouring reticular neurons allowed experimental differentiation between different types of central respiratory reflex actions. In experiments on anaesthetized dogs the responses to chemoreceptor and baroreceptor excitation and to pulmonary inflation were investigated. Chemoreceptor excitation leads to activation of inspiratory, expiratory, and reticular neurons, whereas the baroreceptor afferents act in the opposite direction. In contrast moderate lung inflation causes more specific effects: activation of expiratory neurons, inactivation of inspiratory neurons. But if a certain degree of lung inflations is exceeded a more general inhibition of both inspiratory and expiratory neurons takes place. These results only apply to the "typical" respiratory neurons. The principles used to distinguish between the different types of reflexes are proposed for a basis of classification also of other neural, chemical or pharmacological influences on breathing.