Intra-cardiac transfer of fatty acids from capillary to cardiomyocyte.

Blood-borne fatty acids (Fa) are important substrates for energy conversion in the mammalian heart. After release from plasma albumin, Fa traverse the endothelium and the interstitial compartment to cross the sarcolemma prior to oxidation in the cardiomyocytal mitochondria. The aims of the present study were to elucidate the site with lowest Fa permeability (i.e., highest Fa resistance) in the overall Fa trajectory from capillary to cardiomyocyte and the relative contribution of unbound Fa (detach pathway, characterized by the dissociation time constant τAlbFa) and albumin-bound Fa (contact pathway, characterized by the membrane reaction rate parameter dAlb) in delivering Fa to the cellular membranes. In this study, an extensive set of 34 multiple indicator dilution experiments with radiolabeled albumin and palmitate on isolated rabbit hearts was analysed by means of a previously developed mathematical model of Fa transfer dynamics. In these experiments, the ratio of the concentration of palmitate to albumin was set at 0.91. The analysis shows that total cardiac Fa permeability, Ptot, is indeed related to the albumin concentration in the extracellular compartment as predicted by the mathematical model. The analysis also reveals that the lowest permeability may reside in the boundary zones containing albumin in the microvascular and interstitial compartment. However, the permeability of the endothelial cytoplasm, Pec, may influence overall Fa permeability, Ptot, as well. The model analysis predicts that the most likely value of τAlbFa ranges from about 200 to 400 ms. In case τAlbFa is fast, i.e., about 200 ms, the extracellular contact pathway appears to be of minor importance in delivering Fa to the cell membrane. If Fa dissociation from albumin is slower, e.g. τAlbFa equals 400 ms, the contribution of the contact pathway may vary from minimal (dAlb≤5 nm) to substantial (dAlb about 100 nm). In the latter case, the permeability of the endothelial cytoplasm varies from infinite (no hindrance) to low (substantial hindrance) to keep the overall Fa flux at a fixed level. Definitive estimation of the impact of endothelial permeability on Ptot and the precise contribution of the contact pathway to overall transfer of Fa in boundary zones containing albumin requires adequate physicochemical experimentation to delineate the true value of, among others, τAlbFa, under physiologically relevant circumstances. Our analysis also implies that concentration differences of unbound Fa are the driving force of intra-cardiac Fa transfer; an active, energy requiring transport mechanism is not necessarily involved. Membrane-associated proteins may facilitate Fa transfer in the boundary zones containing albumin by modulating the membrane reaction rate parameter, dAlb, and, hence, the contribution of the contact pathway to intra-cardiac Fa transfer.

Dimensions of compartments and membrane surfaces in the intact rabbit heart of importance in studies on intramyocardial transfer of blood-borne substances.

Cardiac studies on the uptake, storage and intramyocardial transfer of blood-borne substances require detailed information on the geometric ultrastructural dimensions of myocardial compartments and parts thereof, and the membranes separating these compartments. Such a specific ultrastructural set of data of the heart is yet lacking. In the present study, we quantitatively assessed these dimensions in glutaraldehyde-perfusion fixed rabbit hearts by means of histological and tailored mathematical techniques. We showed the true ellipsoid nature of the myocardial capillary cross section and estimated the mean capillary diameter dcap. After correction for the ellipsoid shape, dcap was found to be 5.21±1.41 µm. Effective widths of the endothelial cell and the pericapillary interstitium (is1), dimensions of importance in diffusion, amounted to 187±7 and 160±10 nm, respectively. The fractional volume of the large vessels (arteries and veins larger than 10 µm), capillaries, endothelium, is1, cardiomyocytes, non-pericapillary interstitium is2, t-tubular compartment and interstitial cells amounted on average to 5.92%, 9.36%, 1.83%, 1.94%, 73.07%, 5.97%, 0.95% and 0.96%, respectively, of total myocardial volume, defined as the cardiac tissue volume, the large blood vessels included. Normalized to total myocardial volume, the surface area of the luminal and abluminal endothelial membranes and of the cardiomyocyte membrane opposing the endothelial cells amounted to 75.2±5.5·10³, 82.2±6.0·10³ and 89.1±6.5·10³ m²/m³, respectively. The present study provides quantitative information about ultrastructural dimensions of the adult rabbit heart, among others, of importance for studies on cardiac uptake, and intramyocardial transfer and storage of blood-supplied substances.

Modeling Fatty Acid Transfer from Artery to Cardiomyocyte.

Despite the importance of oxidation of blood-borne long-chain fatty acids (Fa) in the cardiomyocytes for contractile energy of the heart, the mechanisms underlying the transfer of Fa from the coronary plasma to the cardiomyocyte is still incompletely understood. To obtain detailed insight into this transfer process, we designed a novel model of Fa transfer dynamics from coronary plasma through the endothelial cells and interstitium to the cardiomyocyte, applying standard physicochemical principles on diffusion and on the chemical equilibrium of Fa binding to carrier proteins Cp, like albumin in plasma and interstitium and Fatty Acid-Binding Proteins within endothelium and cardiomyocytes. Applying these principles, the present model strongly suggests that in the heart, binding and release of Fa to and from Cp in the aqueous border zones on both sides of the cell membranes form the major hindrance to Fa transfer. Although often considered, the membrane itself appears not to be a significant hindrance to diffusion of Fa. Proteins, residing in the cellular membrane, may facilitate transfer of Fa between Cp and membrane. The model is suited to simulate multiple tracer dilution experiments performed on isolated rabbit hearts administrating albumin and Fa as tracer substances into the coronary arterial perfusion line. Using parameter values on myocardial ultrastructure and physicochemical properties of Fa and Cp as reported in literature, simulated washout curves appear to be similar to the experimentally determined ones. We conclude therefore that the model is realistic and, hence, can be considered as a useful tool to better understand Fa transfer by evaluation of experimentally determined tracer washout curves.

Echogenic perfluorohexane-loaded macrophages adhere in vivo to activated vascular endothelium in mice, an explorative study.

BACKGROUND: Macrophages may concentrate ultrasound contrast agents and exhibit selective adhesion to activated endothelium. The present study investigates in mice the potential of perfluorohexane (PFH) loaded macrophages to act as ultrasound contrast agent with high reflectivity and specifically targeted at (atherosclerotic) vascular lesions. METHODS: Lung passage was evaluated with a mouse echo scanner after injection, at a slow pace or as a bolus, of varying doses of PFH-loaded and unloaded bone marrow macrophages (BMM) into the jugular vein. The interaction of PFH-loaded and unloaded BMM with TNF-α stimulated carotid artery endothelium after tail vein injection was assessed by means of intravital microscopy. RESULTS: High doses of jugular vein injected PFH-loaded BMM were visible with ultrasound in the pulmonary artery and detectable in the carotid artery. At intravital microscopy, tail vein injected BMM exhibited rolling and adhesion behavior at the TNF-α stimulated carotid endothelium, similar to that of native blood leukocytes. Rolling behavior was not different between PFH-loaded and unloaded BMM (p = 0.38). CONCLUSION: In vivo, perfluorohexane loaded macrophages pass the pulmonary circulation and appear on the arterial side. Moreover, they roll and adhere selectively to activated endothelium under physiological flow conditions. These findings indicate that perfluorohexane loaded BMM could be used to study processes in vivo where endothelial activation plays a role, such as atherosclerosis.

Arterial stiffness and hypertension in a large population of untreated individuals: the Rotterdam Study.

OBJECTIVE: We studied whether arterial stiffness measured as aortic pulse wave velocity (aPWV) and carotid distensibility was associated with different subtypes of hypertension in a large population of untreated middle-aged and elderly men and women. METHODS: The study was conducted within the framework of the population-based Rotterdam Study. We included 4088 individuals with information on aPWV, with 3554 individuals with carotid distensibility measurements without use of antihypertensive medication. Isolated systolic hypertension (ISH) was defined as SBP at least 140 mmHg and DBP less than 90 mmHg. Combined systolic and diastolic hypertension (Sys/Dia hypertension) was defined as SBP at least 140 mmHg and DBP at least 90 mmHg. Analysis of covariance was used to compare means of arterial stiffness for the different subtypes of hypertension. Multinomial logistic regression analysis was performed to investigate the association of arterial stiffness and the subtypes of hypertension in models adjusted for age, sex, mean arterial pressure, heart rate and cardiovascular risk factors. RESULTS: The mean age of the individuals was 68 years: 45.3% were men, 1597 individuals had ISH and 441 individuals had Sys/Dia hypertension. aPWV was higher (13.2 vs. 12.9 m/s; P = 0.008) in individuals with ISH compared to those with Sys/Dia hypertension. Multivariate odds ratios and corresponding 95% confidence interval of aPWV for ISH were 1.53 (1.38-1.71) and 1.28 (1.09-1.53) for Sys/Dia hypertension. Corresponding odds ratios associated with carotid distensibility were 0.84 (0.75-0.94) and 0.66 (0.54-0.81), respectively. Age significantly modified the association of aPWV with subtypes of hypertension (P < 0.001). CONCLUSION: In a large untreated population, we found significant associations of both aPWV and carotid distensibility with ISH and Sys/Dia hypertension. individuals with ISH had higher values of aortic stiffness when compared to individuals with Sys/Dia hypertension, a difference that was most pronounced at older age. The results suggest that aortic stiffness contributes to ISH in older individuals without treatment for hypertension.

Common carotid intima-media thickness in cardiovascular risk stratification of older people: the Rotterdam Study.

AIM: Non-invasive measures of atherosclerosis, such as carotid intima-media thickness (cIMT), may improve global cardiovascular risk prediction. The aim of this study was to determine whether common carotid IMT in addition to traditional risk factors improves risk classification in a general population of older people. METHODS AND RESULTS: A group of 3580 non-diabetic people aged 55-75 years and free of cardiovascular disease at baseline were followed for a median time of 12.2 years. Compared to models based on Framingham risk factors, we studied the ability of common cIMT measurement to better classify people into categories of low (<10%), intermediate (10-20%) and high (>20%) 10-year risk of hard coronary heart disease (CHD) and stroke. In older men, addition of cIMT to Framingham risk factors did not improve prediction of hard CHD or stroke. In older women, addition of cIMT to Framingham risk factors significantly improved risk classification. cIMT improved the C-statistic of the model for hard CHD from 0.711 to 0.719 and for stroke from 0.712 to 0.721, at good calibration. Reclassification was least in the majority of women classified as low risk (4% (n = 76) for hard CHD and 3% (n = 62) for stroke) and most substantial in women at intermediate risk (43% (n = 70) for hard CHD and 28% (n = 76) for stroke). The net reclassification improvement in women was 8.2% (p = 0.03) for hard CHD and 8.0% (p = 0.06) for stroke. CONCLUSION: cIMT had some additional value beyond traditional risk factors in the cardiovascular risk stratification of older women, but not of older men.

The change in arterial stiffness over the cardiac cycle rather than diastolic stiffness is independently associated with left ventricular mass index in healthy middle-aged individuals.

BACKGROUND: The current standard for arterial stiffness assessment, aortic pulse wave velocity (aPWV), is measured at diastolic pressure. Arterial stiffness, however, is pressure dependent. At the carotid artery level, the degree of this dependency can be quantified as the difference (ΔPWV) between systolic and diastolic (cPWVd) carotid pulse wave velocity. Biomechanically, a greater ΔPWV implies greater increases in left ventricular afterload with physical activity. Therefore, we hypothesized, that ΔPWV is more strongly associated with left ventricular mass index (LVMI) than aPWV and cPWVd. METHODS: In 1776 healthy individuals from the Asklepios cohort (age 35-55 years), ΔPWV was obtained from combined carotid artery ultrasound and tonometry recordings. Multiple linear regression analysis was performed to investigate the associations of ΔPWV, cPWVd and aPWV with LVMI, adjusting for age, sex, mean blood pressure (MBP), central pulse pressure, and other possible confounders. RESULTS: ΔPWV was 2.4 ±â€Š1.2 m/s (mean ±â€ŠSD), ranging from 0.8 m/s, indicating almost constant arterial stiffness over the cardiac cycle, to 4.4 m/s, reflecting substantial pressure dependency. ΔPWV was significantly associated with LVMI (ß of 2.46 g/m per m/s, P < 0.001), even after full adjustment (ß of 0.56 g/m per m/s, P = 0.03). cPWVd and aPWV had clear crude associations with LVMI (P < 0.001), but lost significance after adjustment (ß of -0.48 and -0.33 g/m per m/s, with P = 0.11 and 0.2, respectively). CONCLUSION: The change in arterial stiffness over the cardiac cycle, rather than diastolic stiffness, is independently associated with LVMI in healthy middle-aged individuals. Therefore, the pressure dependency of arterial stiffness should be considered in cardiovascular risk assessment.

Spatial distension variations are associated with focal atherosclerotic plaques.

BACKGROUND: Alterations in arterial absolute distension (AD) and relative distension (RD) in the common carotid artery (CCA), as observed by ultrasound, may relate to atherosclerotic processes in a nearby vascular segment. The main objective of the study was to establish to what extent these mechanical vascular alterations relate to carotid bulb stenosis. METHODS: The AD, RD and their interrecording variation (Delta AD and Delta RD), representing the spatial inhomogeneity of AD and RD, were evaluated in 308 CCA and associated with the degree of bulb stenosis. RESULTS: The mechanical CCA parameters were marginally correlated to the degree of ipsilateral (r = -0.14 for AD and r = -0.28 for RD) and contralateral stenosis (r = -0.21 for AD and r = -0.27 for RD). However, Delta AD (r = 0.54; p < 0.001) and Delta RD (r = 0.59; p < 0.001) were significantly correlated to ipsilateral stenosis, independent of age, gender and pulse pressure. CONCLUSIONS: The indices of CCA arterial stiffness are weakly associated with ipsilateral bulb stenosis, but their interrecording variation is a good indicator for atherosclerotic alterations in the carotid bulb. The results indicate that a focal atherosclerotic lesion is associated with modifications in the dynamic characteristics of a nearby vascular segment.

Critical appraisal of targeted ultrasound contrast agents for molecular imaging in large arteries.

Molecular imaging may provide new insights into the early detection and development of atherosclerosis before first symptoms occur. One of the techniques in use employs noninvasive ultrasound. In the past decade, experimental and clinical validation studies showed that for the microcirculation targeted ultrasound contrast agents, such as echogenic liposomes, microbubbles and perfluorocarbon emulsions, do improve visualization of specific structures. For large arteries, however, successful application is less obvious. In this review, we will address the challenges for molecular imaging of large arteries. We will discuss the problems encountered in the use of targeted ultrasound contrast agents presently available, mainly based on data obtained in flow chambers and animal studies because clinical studies are lacking. We conclude that molecular imaging of activated endothelium in large- and middle-sized arteries by site-specific accumulation of contrast material is still difficult to achieve due to wall shear stress conditions in these vessels.

Noninvasive assessment of arterial stiffness should discriminate between systolic and diastolic pressure ranges.

Arterial stiffening plays an important role in the development of hypertension and cardiovascular diseases. The intrinsically nonlinear (ie, pressure-dependent) elastic behavior of arteries may have serious consequences for the accuracy and interpretation of arterial stiffness measurements and, ultimately, for individual patient management. We determined aortic pressure and common carotid artery diameter waveforms in 21 patients undergoing cardiac catheterization. The individual pressure-area curves were described using a dual exponential analytic model facilitating noise-free calculation of incremental pulse wave velocity. In addition, compliance coefficients were calculated separately in the diastolic and systolic pressure ranges, only using diastolic, dicrotic notch, and systolic data points, which can be determined noninvasively. Pulse wave velocity at systolic pressure exhibited a much stronger positive correlation with pulse pressure (P<0.001) and age (P=0.012) than pulse wave velocity at diastolic pressure. Patients with an elevated systolic blood pressure (>140 mm Hg) had a 2.5-times lower compliance coefficient in the systolic pressure range than patients with systolic blood pressures <140 mm Hg (P=0.002). Most importantly, some individuals, with comparable age or pulse pressure, had similar diastolic but discriminately different systolic pulse wave velocities and compliance coefficients. We conclude that noninvasive assessment of arterial stiffness could and should discriminate between systolic and diastolic pressure ranges to more precisely characterize arterial function in individual patients.

Genetic variation in the renin-angiotensin system and arterial stiffness. The Rotterdam Study.

We studied the associations of three renin-angiotensin system polymorphisms, angiotensin-converting enzyme (ACE) I/D, angiotensinogen 235 M/T, and angiotensin II receptor type I 573 C/T, with arterial stiffness. The study was embedded in the Rotterdam Study, a population-based study older adults. The association of the polymorphisms with pulse wave velocity, the carotid distensibility, and pulse pressure was investigated in 3706 subjects. We found no association of the ACE I/D polymorphism with pulse wave velocity, but the D-allele was associated with a lower distensibility coefficient (p = 0.05) and higher pulse pressure (p = 0.01). For the angiotensinogen 235 M/T polymorphism, no significant associations with either pulse wave velocity (p = 0.71), the distensibility coefficient (p = 0.16) or pulse pressure (p = 0.34) were found. Also, we found no significant associations of pulse wave velocity (PWV) (p = 0.32), the distensibility coefficient (p = 0.08), and pulse pressure (p = 0.09) with the angiotensin II receptor type 1 573 C/T polymorphism. No epistatic effects were observed between the three renin-angiotensin system (RAS) genes with arterial stiffness. Our findings suggest that genetic variation in the renin-angiotensin system may play a role in determining carotid distensibility and pulse pressure.

The dicrotic notch as alternative time-reference point to measure local pulse wave velocity in the carotid artery by means of ultrasonography.

OBJECTIVES: Increased arterial stiffness is associated with cardiovascular disease. Its applicability in individual patient management, however, is limited due to lack of reliable methods. We developed a method to measure arterial stiffness by means of local pulse wave velocity (PWV), using multiple M-mode ultrasound and the dicrotic notch (PWVdn) rather than the systolic foot (PWVsf) as time-reference point. METHODS: Systolic foot and dicrotic notch were determined in 14 simultaneously recorded distension waveforms obtained in young and older participants (mean age 26 and 59 years). Linear regression was performed on echo-line position and time-reference point, resulting in a local PWV estimate, either PWVsf or PWVdn. RESULTS: PWVdn, at about mean arterial pressure, had a better intra-individual variability (0.6 m/s) than PWVsf (1.1 m/s). The expected difference in stiffness between the two age categories was identified by PWVdn (P < 0.0001), but not by PWVsf. Moreover, in contrast to PWVsf, PWVdn showed a significant correlation with relative distension (r = 0.56) and the local distensibility coefficient (r = 0.52). CONCLUSION: PWVdn is a noninvasive and suitable measure of arterial stiffness: it has a good reproducibility, discriminates well between age groups, and correlates with local distensibility. PWVdn does not require additional assessment of distance or local pulse pressure. Furthermore, PWVdn is measured locally, at near-mean arterial pressure, thereby better reflecting the effective arterial stiffness, which determines the load the left ventricle is subjected to as it ejects blood.

Genetic variation in the fibrinogen-alpha and fibrinogen-gamma genes in relation to arterial stiffness: the Rotterdam Study.

OBJECTIVE: Arterial stiffness increases with age and predicts cardiovascular disease. Fibrinogen is an acute-phase protein and some studies showed an association with arterial stiffness. We studied genetic variation in the fibrinogen-alpha (FGA) and fibrinogen-gamma (FGG) genes, by means of single nucleotide polymorphisms (FGA: -58 G/A, 1374 G/A, 1526 T/C, 312 Thr/Ala, and FGG: 4288 G/A, 6326 G/A, 7792 T/C) and resultant haplotypes in relation to arterial stiffness. METHODS: The present study (n = 3891) was embedded in the Rotterdam Study. Associations of the fibrinogen level, genotypes and haplotypes with aortic stiffness (pulse wave velocity), carotid stiffness (distensibility coefficient) and pulse pressure were investigated in men and women by analyses of variance, linear regression and by haplotype analyses. Analyses were adjusted for age, mean arterial pressure, heart rate, known cardiovascular risk factors and atherosclerosis. RESULTS: Genotype analyses yielded associations of FGA-58 G/A (P = 0.040, for trend) and FGA-1526 T/C (P = 0.004, for trend) with the fibrinogen levels, but no consistent associations with arterial stiffness, in women. FGA-haplotype 4 was associated with the fibrinogen level (P = 0.02) in women. FGA-haplotype 3 and FGG-haplotype 2 were associated with aortic stiffness (P = 0.05) in women. No associations were found in men. CONCLUSION: Findings indicate that the fibrinogen level and genetic variation in the FGA and FGG genes may influence arterial stiffness in women.

Wall irregularity rather than intima-media thickness is associated with nearby atherosclerosis.

In addition to intima-media thickness (IMT), IMT inhomogeneity may carry information about atherosclerosis progression. In 147 vascular diseased patients (mean 66 y, 48% male), we determined the carotid bulb stenosis degree based on local Doppler blood flow velocities. Common carotid artery (CCA) morphologic characteristics, i.e. IMT, IMT-inhomogeneity (intraregistration variation) and IMT uni- and bilateral intrasubject variation (DeltaIMT), were measured using multiple M-mode. Associations of morphologic characteristics, stenosis degree and Framingham score were evaluated with Pearson correlation (r) and multiple regression analysis. The IMT distributions for subjects without and with stenosis were not similar. The stenosis degree score correlated significantly to unilateral (r=0.68) and bilateral DeltaIMT (r=0.62), IMT (r=0.41) and IMT-inhomogeneity (r=0.45). The averaged IMT and IMT-inhomogeneity increased slightly for singular stenosis and abruptly for multiple stenoses. Mean uni- and bilateral DeltaIMT per stenosis degree increased linearly with this degree, reaching a correlation close to 1 (r=0.98 and r=0.97). Interestingly, the majority of the subjects with a moderate to severe bulb stenosis exhibited a carotid IMT lower than the considered critical threshold of 0.9 mm. In conclusion, although CCA is not prone to plaques, its morphologic characteristics are positively correlated with stenosis degree score and other risk scores. DeltaIMT can be more reliable derived from inter-registration rather than from intra-registration variation. In the CCA, DeltaIMT substantiates vascular alteration better than IMT.

Contribution of vascular and neural segments to baroreflex sensitivity in response to postural stress.

BACKGROUND/AIMS: The baroreflex pathway has a vascular and a neural segment, both being modulated by variations in peripheral blood pressure (BP). Besides overall baroreceptor sensitivity (BRS), defined as the spectral relationship between changes in peripheral BP and R-R interval within the frequency band of 0.05-0.15 Hz, vascular and neural segment contributions to the overall BRS can be distinguished. We test the hypothesis that changes in overall BRS following a postural maneuver mainly originate from the vascular (peripheral pressure to carotid artery diameter) rather than the neural segment (carotid artery diameter to R-R interval). METHODS: Peripheral pressure (Finapress), carotid artery diameter (ultrasound in B-/M-mode) and electrocardiogram values of 20 young subjects in supine and upright-seated postures were recorded simultaneously. Transfer gains were computed for the segmental and overall responses. RESULTS: Postural change significantly increases peripheral BP and carotid artery diameter. The vascular segment has a uniform spectral distribution. Statistical analyses revealed that postural change decreased overall (p < 0.004) and vascular (p < 0.0001) transfer gains, but did not modify neural gain. CONCLUSIONS: Unlike the neural segment, the vascular segment is frequency non-specific. The decrease in overall BRS due to a postural change is mainly explained by the reduced transfer gain of the vascular segment.

The interleukin-6-174 G/C promoter polymorphism and arterial stiffness; the Rotterdam Study.

Arterial stiffness normally increases with age and has been established as a precursor of cardiovascular disease. Interleukin-6 is a pleiotropic inflammatory cytokine with an important role in the inflammatory cascade, such as up-regulation of C-reactive protein (CRP). The interleukin-6-174-G/C promoter polymorphism appears to influence levels of inflammatory markers, which have been shown to be associated with arterial stiffness. We studied the association of this polymorphism with levels of interleukin-6 and CRP and with arterial stiffness. The study (n=3849) was embedded in the Rotterdam Study, a prospective, population-based study. Analyses on the association between the -174-G/C polymorphism and pulse wave velocity, distensibility coefficient, and pulse pressure were performed using analyses of variance. Analyses on the levels of inflammatory markers and arterial stiffness were performed using linear regression analyses. Analyses were adjusted for age, sex, mean arterial pressure, heart rate, known cardiovascular risk factors, and atherosclerosis. We found pulse wave velocity to be 0.35 m/s higher for CC-homozygotes vs. wildtype GG-homozygotes (p = 0.018) with evidence for an allele-dose effect (p trend = 0.013), and a similar pattern for pulse pressure (p trend = 0.041). No apparent consistent association with the distensibility coefficient was found. CRP levels were associated with pulse wave velocity (p = 0.007). In conclusion, the interleukin-6-174 G/C polymorphism is associated with increased arterial stiffness and pulse pressure.