Robert Loncar

Spatial heterogeneity of blood flow in the dog heart. I. Glucose uptake, free adenosine and oxidative/glycolytic enzyme activity.

The spatial heterogeneity of myocardial perfusion and metabolism was studied in 11 anaesthetized dogs under resting conditions. In each heart local myocardial blood flow was assessed using the tracer microsphere technique in 256 samples (mean mass: 83.1 mg) taken from the left anterior ventricular wall. In the same samples, the following biochemical parameters were determined: accumulation of [3H]-deoxyglucose (a measure of glucose uptake), free cytosolic adenosine (S-adenosylhomocysteine accumulation technique, a measure of tissue oxygenation and a possible mediator of blood flow regulation), and the specific activities of oxidative (citrate synthase, cytochrome-c-oxidase) and glycolytic (hexokinase, phosphoglycerate kinase) enzymes. Capillary density and mitochondrial and myofibril volume densities were determined by morphometry. Myocardial perfusion in each sample (average 0.77 ml min−1 g−1) varied between 0.1 and 2.5 times the mean (coefficient of variation 0.30±0.02). [3H]-deoxyglucose was deposited locally in proportion to perfusion. Samples showing low flow (< 0.2 ml min−1 g−1) did not exhibit increased levels of cytosolic adenosine. The specific activities of the oxidative and glycolytic enzymes, however, were uniformly distributed between low and high flow areas. Furthermore, capillary density and mitochondrial and myofibril densities were similar in high and low flow regions. The results show firstly that local glucose metabolism in the heart occurs in proportion to local blood flow, suggesting that high flow regions have a higher than average metabolic rate. Secondly, regions of low flow are not compromized by critical oxygenation and most likely have a lower than average oxygen demand and finally, the homogeneous distribution of oxidative and glycolytic enzymes, as well as the homogeneous myocardial ultrastructure, suggest that areas with high and low blood flow under resting conditions may increase their metabolic rate to similar levels when required.

Coronary Reserve of High- and Low-Flow Regions in the Dog Heart Left Ventricle

BACKGROUND Left ventricular myocardial blood flow is spatially heterogeneous. The hypothesis we tested was whether myocardial areas with a steady-state flow <0.5 times mean flow are underperfused and areas with flow > 1.5 times mean flow are overperfused. METHODS AND RESULTS In anesthetized beagle dogs (n=10), the relationship between local blood flow versus S-adenosylhomocysteine (SAH) concentration, a measure of the free intracellular adenosine concentration, and lactate, a measure of the myocardial NADH/NAD+ ratio, were determined under control conditions and after coronary constriction. Control local myocardial blood flow was 0.99+/-0.46 mL x min(-1) x g(-1), with a coefficient of variation of 0.36+/-0.12 (n=256 per heart; sample wet mass, 125+/-30 mg). Tissue concentrations of SAH (3.4+/-2.5 nmol/g) and lactate (1.88+/-0.80 micromol/g) were not elevated in low-flow samples. However, after coronary artery constriction, poststenotic blood flow decreased from 1.00+/-0.27 to 0.49+/-0.22 mL x min(-1) x g(-1) (P<0.04), with significant correlation between local SAH and flow (r=-0.59) and lactate and flow (r = -0.50). Although nearly all samples from control high-flow regions showed increased SAH concentrations if relative flow after stenosis was <1.0, control low-flow samples frequently displayed low SAH concentrations. The percent reduction in flow determined the changes in the local SAH and lactate concentration, independent of the local control blood flow. CONCLUSIONS When the coronary inflow is unrestricted, the oxygen supply to control low-flow regions meets metabolic demand. Flow to control high-flow regions reflects a higher local demand rather than overperfusion. Thus, blood flow heterogeneity most likely reflects differences in aerobic metabolism.

Fatal bleeding due to a heparin-like anticoagulant in a 37-year-old woman suffering from systemic mastocytosis.

A 37-year-old female patient with systemic mastocytosis who was admitted with severe unexplained bleeding symptoms is studied. Laboratory procedures established the diagnosis of a patient-derived—heparin-like anticoagulant as a very rare hemostatic abnormality predisposing to bleeding. The patient died from refractory disease despite therapy with protamine, initiation of chemotherapy, and supportive measures. The case illustrates the clinical presentation and diagnosis of heparin-like anticoagulants. Etiology, pathophysiology, and therapeutic options are discussed.

Heterogeneity of metabolic parameters in the left ventricular myocardium and its relation to local blood flow

Abstract It is well established that myocardial blood flow is heterogeneous on the local level. During recent years comprehensive studies have been undertaken to assess the relation between myocardial metabolism and spatial blood flow heterogeneity. Based on the type of measurements two major groups of studies have been performed: enzyme activity and tissue metabolite level assessments. Enzyme activity measurements have provided only limited insight into the coupling of local metabolism and flow. This is probably due to the fact that, in addition to estimated Vmax values, local substrate affinity (Km values) and substrate concentrations affect the metabolite fluxes. However, the latter two variables remain normally unknown. In contrast, valuable insight has been obtained concerning flow-metabolism matching from tissue metabolite measurements, especially when connected with mathematical model analyses. The latter permitted the calculation of metabolic flux rates (e.g., production of oxidation water, citric acid cycle flux, glucose uptake, fatty acid uptake) or the translation of the metabolic indexes into physiologically meaningful local metabolite concentrations (e.g., free cytosolic adenosine). The bottom line of the studies reported to date is that the broad range of myocardial flows observed under resting control conditions correlates with local metabolism possibly affected by spatial differences in adrenergic stimulation. Thus, high flow samples exhibit a higher oxidative metabolism than low flow samples. As a result the flow threshold below which local myocardial ischemia ensues is higher in control high flow samples. The importance of these findings with respect to local flow-metabolism matching is underlined by the finding that the probability of developing an infarction following ischemia/reperfusion is related to the functional state of the myocardium under control conditions, i.e., the local level of flow-metabolism matching.

HPA-1 polymorphism of αIIbβ3 modulates platelet adhesion onto immobilized fibrinogen in an in-vitro flow system

BackgroundPlatelet adhesion and subsequent thrombus formation on a subendothelial matrix at the site of vascular damage play a crucial role in the arrest of posttraumatic bleeding but also in different pathological thrombotic events, such as acute coronary syndrome and stroke. Recently published studies have clearly demonstrated that platelet integri αIIbβ3 is intimately involved in the occlusive thrombus formation at the site of endothelial damage. Therefore, any genetic variation in the expression of this receptor may lead to an excessive bleeding or excessive thrombus formation. In this study, we evaluated the influence of HPA-1 polymorphism of integrin αIIbβ3 on platelet adhesion onto immobilized fibrinogen using an in vitro system simulating blood flow.MethodsPlatelets in anticoagulated whole blood [49 healthy previously genotyped blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 50 s-1, 500 s-1 and 1500 s-1). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 sec, 1 and 5 minutes after start of perfusion.ResultsDuring perfusion, the platelet adhesion linearly increased with regard to exposition time and shear rate. Perfusion of blood preincubated with Abciximab over fibrinogen-coated cover-slips showed reduced platelet adherence (absolute fluorescence: 168 ± 35 U vs. 53000 ± 19000 at control experiments, p < 0.05), as well as by perfusion over BSA-coated glass coverslips. Platelet with HPA-1a/1a genotype exhibited initial better adhesion but they also exhibited higher detachment under arterial flow conditions compared to the HPA-1b/1b platelets. Analysis of stable adhesion rate indicate that the platelets carrying the HPA-1b/1b genotype have a higher reactivity threshold for initial interaction with fibrinogen but under the higher shear rate (in regard to time of perfusion) also realize more stable bonds with fibrinogen than platelets with the HPA-1a/1a genotype.ConclusionOur data support the contention that genetically determined variants of platelet integrins αIIbβ3 could play a role in arterial thrombogenesis and thus confirm the hypothesis derived from epidemiological studies.

Platelet adhesion onto immobilized fibrinogen under arterial and venous in-vitro flow conditions does not significantly differ between men and women.

BackgroundGender-related differences in incidence of arterial thrombosis have been a focus of interest for years. The platelet integrin αIIbβ3 is primarily responsible for the interaction between platelets and fibrinogen and consecutive thrombus growth. In this study, we evaluated platelet adhesion onto immobilized fibrinogen under venous and arterial flow conditions in men and women.MethodsPlatelets in whole anticoagulated blood were labelled with the fluorescence dye Mepacrine and perfused through the rectangular flow chamber over glass cover slips coated with fibrinogen (shear rates of 50 s-1, 500 s-1 and 1500 s-1). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 seconds, 1 and 5 minutes after the start of perfusion.ResultsDuring perfusion, the platelet adhesion linearly increased in regard to exposition time and shear rate. After five minutes of perfusion the platelet adhesion onto immobilized fibrinogen showed no significant gender related difference, neither at 50 s-1 nor at 500 s-1 and 1500 s-1 (p > 0.05), respectively. No significant difference in platelet adhesion onto immobilized fibrinogen, in regard to the menopausal status, was either observed (p > 0.05).ConclusionIn our in vitro experimental system, hormonal differences between men and women did not influence platelet adhesion onto immobilized fibrinogen, neither under venous nor under arterial rheological conditions.

Screening of plasma homocysteine in peripheral arterial disease

BACKGROUND. Homocysteine (HCY) was recently established as an independent risk factor for atherosclerosis. The prevalence of an increased homocysteine plasma concentration was reported to be up to 6-fold higher in patients with different locations of arterial occlusive diseases. AIM. This study evaluated critically whether the total HCY plasma concentration can be used as a screening marker for peripheral arterial disease in the general population. METHODS. Study subjects were 40 patients (51.8 ± 7.5 years) with symptomatic lower limb peripheral arterial disease (PAD) (stage II) and 40 healthy volunteers (45.6 ± 6.8 years, P < 0.05 vs PAD). The percentage of women in both groups was 30%. The plasma HCY concentration was determined by using derivatization techniques and subsequent fluorescence high-performance liquid chromatography. RESULTS. Total plasma HCY concentration was significantly higher in the PAD group than in controls (14.90 ± 5.78 μM vs 11.32 ± 2.95 μM, respectively, P < 0.001). Also, the coefficient of variation of plasma HCY in PAD was significantly higher than that in the control group, 0.38 vs 0.25 (P < 0.001), respectively, reflecting greater interindividual differences. In addition to a PAD-specific effect, the plasma HCY concentration was also dependent on gender and age (both P < 0.05). Sensitivity and specificity of HCY as a marker of PAD were 0.3 and 0.95, respectively. Positive and negative predictive values were 0.85 and 0.42, respectively. CONCLUSIONS. From these data it is concluded that HCY metabolism may have an influence on the development of PAD in one-third of all patients with PAD, and that total plasma HCY concentration may not be suited as a screening test for PAD in the general population but rather serves as a monitoring marker in certain risk groups.

Effects of homocysteine on vascular and tissue adenosine: a stake in homocysteine pathogenicity?

Abstract Homocysteine may have deleterious effects on the cardiovascular system. It has been hypothesized that these effects may be brought about by a decrease in the adenosine concentration via the S-adenosylhomocysteine hydrolase reaction. A requirement for this causal relationship is proof of a reduction in vascular adenosine concentration during conditions of elevated homocysteine concentrations. In the present communication we summarize published data obtained during systematic variation of the arterial homocysteine concentration. Most of the results reported show that an increase in homocysteine concentration to 100μM is associated with a 20–50% decrease in vascular adenosine concentration and an increase in tissue S-adenosylhomocysteine level. Homocysteine effects on the adenosine concentration seem to be more pronounced under conditions of impaired oxygenation. Further experiments, in particular on organs and tissue that release high amounts of homocysteine, i.e., the liver, are warranted to study the potential effects of homocysteine on vascular and tissue adenosine concentrations and consequent effects on organ function. The evidence obtained may be relevant for future assessment of risk indicators in conjunction with homocysteine pathogenicity, which might potentially be extended to measurements of adenosine or S-adenosylhomocysteine levels.

The TT Genotype of the C677T Polymorphism in the Methylentetrahydrofolate Reductase as a Risk Factor in Thrombotic Microangiopathies: Results From a Pilot Study

In this study, we assessed the potential role of the TT genotype of the gene of the methylenetetrahydrofolate reductase for the manifestation of thrombotic microangiopathies, enrolling 40 affected patients (mean age [± standard deviation] 35 ± 11 years). As a result, the methylenetetrahydrofolate reductase 677TT genotype was more prevalent in patients with thrombotic microangiopathies compared with controls (adjusted odds ratio = 2.58, 95% confidence interval = 1.2-5.7, P = .018), particularly in those suffering from the hemolytic uremic syndrome. A hemolytic more severe clinical course of thrombotic microangiopathies in carriers of the methylenetetrahydrofolate reductase 677TT genotype was not observed. In summary, our findings suggest a significant influence of the methylenetetrahydrofolate reductase genotype on the manifestation of thrombotic microangiopathies. The 677 TT genotype of this polymorphism appears to be a risk factor for manifestation of these rare thrombotic disorders, possibly explained by endothelial activation and increased oxidative stress.

The G1691A Mutation of the Factor V Gene (Factor V Leiden) and the G20210A Mutation of the Prothrombin Gene as Risk Factors in Thrombotic Microangiopathies

Factor V Leiden (FVL) mutation and prothrombin G20210A mutation are common hereditary risk factors for venous thrombosis. In the current study, 40 patients (mean age ± standard deviation, 35 ± 11 years) and 764 healthy control subjects (mean age ± standard deviation, 37 ± 14 years) were enrolled to assess the potential role of these mutations in the manifestation of thrombotic microangiopathies. Compared with controls, neither the heterozygous FVL mutation (7.5% vs 8.5%; P = 1) nor the heterozygous prothrombin mutation (2.5% vs 2.8%; P = 1) was more prevalent in the patients. The findings do not support a significant role of FVL and prothrombin mutations as risk factors for the manifestation of thrombotic microangiopathies. Thus, screening for these mutations does not allow the identification of individuals at increased risk for these rare thrombotic disorders.