Pulmonary hemodynamics and wave reflections in adults with atrial septal defects.

Using high-fidelity micromanometers and flow velocity sensors at right heart catheterization, we compared pulmonary hemodynamics and wave reflections in age-matched normal adults and those with atrial septal defects, separated into three subgroups based on levels of mean pulmonary artery pressure: low (<17 mmHg), intermediate (17-26 mmHg), high (>26 mmHg). We made baseline measurements in all groups and after intravenous sodium nitroprusside in the subgroups. All of the subgroups had higher than normal baseline pulmonary flows and corresponding power that did not differ among the subgroups. The pulmonary vascular resistance, input resistance, and characteristic impedance in the subgroups did not differ from normal. Aside from the elevated flow and power, the hemodynamics in the low subgroup did not differ from normal. The intermediate subgroup had significantly higher than normal right ventricular and pulmonary artery pressures, wave reflections, and shorter wave reflection time, which all reverted to normal after nitroprusside. The high subgroup had similar changes as the intermediate subgroup. Unlike that subgroup, however, the pressures, wave reflections, and reflection return time did not revert to normal after nitroprusside. Hence, elevated wave reflections, but not resistance or characteristic impedance, are the hallmark of pulmonary hypertension in adults with atrial septal defects. Our results demonstrate that detailed measurements of hemodynamics and assessment of responsiveness to vasodilators provide important information about the pulmonary circulation in atrial septal defect. Coupled with studies after defect closure, those results may be a better foundation than current ones for clinical decisions.

Prognostic Utility of Morning Blood Pressure Surge for 20-Year All-Cause and Cardiovascular Mortalities: Results of a Community-Based Study.

BACKGROUND: Morning blood pressure (BP) surge (MS), defined by the MS amplitude, is an independent prognostic factor of cardiovascular outcomes in some, but not all, populations. METHOD AND RESULTS: We enrolled 2020 participants (1029 men; aged 30-79 years) with 24-hour ambulatory BP data. During a median 19.7-year follow-up, 607 deaths (182 by cardiovascular causes) were confirmed from the National Death Registry. The amplitude of sleep-trough MS (STMS) was derived from the difference between morning systolic BP (SBP) and lowest nighttime SBP. The rate of STMS was derived as the slope of linear regression of sequential SBP measures on time intervals within the STMS period. Thresholds for high STMS amplitude and rate were determined by the 95th percentiles (43.7 mm Hg and 11.3 mm Hg/h, respectively). Multivariable Cox models, adjusting for age, sex, body mass index, smoking, alcohol, low-density lipoprotein cholesterol, 24-hour SBP, night:day SBP ratio, and antihypertensive treatment, revealed that a high STMS rate (hazard ratio, 1.666; 95% confidence interval, 1.185-2.341), but not STMS amplitude (hazard ratio, 1.245; 95% confidence interval, 0.984-1.843), was significantly associated with a greater mortality risk. Similarly, STMS rate (hazard ratio, 2.608; 95% confidence interval, 1.554-4.375), but not STMS amplitude, was significantly associated with the risk of cardiovascular mortality (hazard ratio, 0.966; 95% confidence interval, 0.535-1.747). Moreover, the prognostic values of STMS rate were comparable in subjects with or without morning and nocturnal hypertension (P>0.05 for interaction for all). In simulation studies, STMS rate was less susceptible to measurement errors of the sleep-trough SBP than STMS amplitude. CONCLUSIONS: STMS rate could independently help identify subjects with an increased cardiovascular risk.

Hemodynamic Determinants of the Short-Term Blood Pressure Variability: Differential Roles of Arterial Stiffness and Wave Reflection.

BACKGROUND: A high 24-hour ambulatory diastolic (DBP) but not systolic (SBP) blood pressure variability (BPV) is significantly predictive of long-term cardiovascular mortality in untreated hypertensive subjects, independent of office or 24-hour SBP. The present study was aimed to investigate hemodynamic factors that are independently associated with systolic and diastolic BPV from the 24-hour ambulatory blood pressure monitoring (ABPM). METHODS: A cohort of 624 normotensive and 633 untreated hypertensive participants with baseline ABPM was drawn from a community-based survey. BPV was assessed by the read-to-read average real variability of the 24-hour SBP and DBP (ARVs and ARVd, respectively). Hemodynamic variables including total peripheral resistance (TPR), carotid-femoral pulse wave velocity (cf-PWV), and amplitudes of the decomposed forward (Pf) and backward (Pb) carotid pressure waves were analyzed. RESULTS: In multivariable analyses, hemodynamic variables independently associated with 24-hour SBP were 24-hour heart rate (HR), TPR, cf-PWV, Pf, and Pb (model r2 = 0.535). Hemodynamic factors independently associated with ARV were 24-hour HR, Pf, and Pb for ARVs, and 24-hour HR, cf-PWV, Pf, and Pb for ARVd (model R2 = 0.345 and 0.220, respectively). Addition of 24-hour SBP to the ARV models only slightly improved variance explained by the models (R2 = 0.383 and 0.224, respectively). Pb accounted for >50% of total variance of ARVs and ARVd, whereas cf-PWV was a minor determinant of ARVd (<5% of total variance). CONCLUSIONS: ARVd was associated with fewer hemodynamic variables than to 24-hour SBP. Among those hemodynamic variables wave reflection but not arterial stiffness had the dominant independent association with ARV.

Prognostic significance of mechanical biomarkers derived from pulse wave analysis for predicting long-term cardiovascular mortality in two population-based cohorts.

BACKGROUND: Numerous mechanical biomarkers derived from pulse wave analysis (PWA) have been proposed to predict cardiovascular outcomes. However, whether these biomarkers carry independent prognostic value and clinical utility beyond traditional cardiovascular risk factors hasn't been systematically evaluated. We aimed to investigate the additive utility of PWA-derived biomarkers in two independent population-based cohorts. METHODS: PWA on central arterial pressure waveforms obtained from subjects without a prior history of cardiovascular diseases of two studies was conducted based on the wave transmission and reservoir-wave theory: firstly in the Kinmen study (1272 individuals, a median follow-up of 19.8years); and then in the Cardiovascular Disease Risk Factors Two-Township Study (2221 individuals, median follow-up of 10years). The incremental value of the biomarkers was evaluated by net reclassification index (NRI). RESULTS: In multivariate Cox analyses accounting for age, gender, body mass index, systolic blood pressure, fasting glucose, high-density- and low-density-lipoprotein cholesterol, and smoking, only systolic (SC) and diastolic rate constant (DC) of reservoir pressure could independently and consistently predict cardiovascular mortality in both cohorts and the combined cohort (SC: hazard ratio 1.18 [95% confidence interval 1.08-1.28, p<0.001; DC: 1.18 [1.09-1.28], p<0.001]. Risk prediction estimates in traditional risk prediction models were significantly more accurate when incorporating peak of reservoir pressure (NRI=0.049, p=0.0361), SC (NRI=0.043, p=0.0236) and DC (NRI=0.054, p=0.047). CONCLUSIONS: Of all PWA-derived biomarkers, SC and DC were consistently identified as valuable parameters for incremental cardiovascular risk prediction in two large prospective cohorts.

High Short-Term Blood Pressure Variability Predicts Long-Term Cardiovascular Mortality in Untreated Hypertensives But Not in Normotensives.

BACKGROUND: The prognostic value of the short-term blood pressure variability (BPV) from the 24-hour ambulatory blood pressure monitoring (ABPM) remains controversial. The present study aimed to investigate the long-term prognostic value of a high BPV in normotensive and hypertensive subjects from a community-based population. METHODS: A cohort of 624 normotensive and 633 untreated hypertensive Taiwanese participants (overall 669 men, aged 30-79 years) with baseline ABPM and 20-year all-cause and cardiovascular mortality data was drawn from a community-based survey. BPV was assessed by the read-to-read average real variability of the 24-hour diastolic and systolic blood pressure (SBP) (ARVd and ARVs, respectively). RESULTS: In Cox proportional hazards analysis, ARVd predicted cardiovascular mortality independently of office SBP (hazard ratios (HRs) and 95% confidence intervals (CIs) per 1 SD: 1.31 (1.10-1.55), respectively, bivariate analysis), 24-hour SBP (HR: 1.19, 95% CI: 1.00-1.43), and conventional risk factors (age, sex, smoking, total cholesterol, high-density lipoprotein cholesterol, and fasting blood glucose, HR: 1.40, 95% CI: 1.18-1.67). In subjects with hypertension, a high vs. low ARVd (median: 8.8mm Hg) significantly predicted cardiovascular mortality (HR: 2.11, 95% CI: 1.23-3.62 and HR: 2.04, 95% CI: 1.19-3.51, respectively), when the conventional risk factors plus office SBP or 24-hour SBP were accounted for, respectively. Similar but less significant results were obtained with ARVs. A high ARVd or ARVs did not significantly predict cardiovascular mortality in the normotensive subjects. CONCLUSIONS: A high short-term BPV is significantly predictive of long-term cardiovascular mortality in untreated hypertensive but not normotensive community-based subjects, independently of office or 24-hour SBP.

White coat hypertension is more risky than prehypertension: important role of arterial wave reflections.

Arterial aging may link cardiovascular risk to white coat hypertension (WCH). The aims of the present study were to investigate the role of arterial aging in the white coat effect, defined as the difference between office and 24-hour ambulatory systolic blood pressures, and to compare WCH with prehypertension (PH) with respect to target organ damage and long-term cardiovascular mortality. A total of 1257 never-been-treated volunteer subjects from a community-based survey were studied. WCH and PH were defined by office and 24-hour ambulatory blood pressures. Left ventricular mass index, carotid intima-media thickness, estimated glomerular filtration rate, carotid-femoral pulse wave velocity, carotid augmentation index, amplitude of the reflection pressure wave, and 15-year cardiovascular mortality were determined. Subjects with WCH were significantly older and had greater body mass index, blood pressure values, intima-media thickness, carotid-femoral pulse wave velocity, augmentation index, amplitude of the backward pressure wave, and a lower estimated glomerular filtration rate than PH. Amplitude of the backward pressure wave was the most important independent correlate of the white coat effect in multivariate analysis (model r(2)=0.451; partial r(2)/model r(2)=90.5%). WCH had significantly greater cardiovascular mortality than PH (hazard ratio, 2.94; 95% confidence interval, 1.09-7.91), after accounting for age, sex, body mass index, smoking, fasting plasma glucose, and total cholesterol/high-density lipoprotein-cholesterol ratio. Further adjustment of the model for amplitude of the backward pressure wave eliminated the statistical significance of the WCH effect. In conclusion, the white coat effect is mainly caused by arterial aging. WCH carries higher risk for cardiovascular mortality than PH, probably via enhanced wave reflections that accompany arterial aging.

Associations of serum uric acid levels with arterial wave reflections and central systolic blood pressure.

BACKGROUND: Uric acid may be involved in the pathogenesis of hypertension. We investigated the roles of four major hemodynamic parameters of blood pressure, including arterial stiffness, wave reflections, cardiac output (CO), and total peripheral resistance (TPR), in the association between uric acid and central systolic blood pressure (SBP-c). METHODS: A sample of 1303 normotensive and untreated hypertensive Taiwanese participants (595 women, aged 30-79 years) was drawn from a community-based survey. Study subjects' baseline characteristics, biochemical parameters, carotid-femoral pulse wave velocity (cf-PWV), amplitude of the backward pressure wave decomposed from a calibrated tonometry-derived carotid pressure waveform (Pb), CO, TPR, and SBP-c were analyzed. RESULTS: In multi-variate analyses adjusted for age, waist circumference, body mass index, creatinine, total cholesterol, smoking, and heart rate, uric acid significantly correlated with Pb and cf-PWV in men, and Pb and TPR in women. The correlation between uric acid and Pb remained significant in men and women when cf-PWV was further adjusted. In the final multi-variate prediction model (model r(2)=0.839) for SBP-c, the significant independent variables included uric acid (partial r(2)=0.005), Pb (partial r(2)=0.651), cf-PWV (partial r(2)=0.005), CO (partial r(2)=0.062), TPR (partial r(2)=0.021), with adjustment for age, sex, waist circumference, body mass index, creatinine, total cholesterol, smoking, and heart rate. CONCLUSIONS: Uric acid was significantly independently associated with wave reflections, which is the dominant determinant of SBP-c. Uric acid was also significantly associated with SBP-c independently of the major hemodynamic parameters.

Wave reflection and arterial stiffness in the prediction of 15-year all-cause and cardiovascular mortalities: a community-based study.

The value of increased arterial wave reflection, usually assessed by the transit time-dependent augmentation index and augmented pressure (Pa), in the prediction of cardiovascular events may have been underestimated. We investigated whether the transit time-independent measures of reflected wave magnitude predict cardiovascular outcomes independent of arterial stiffness indexed by carotid-femoral pulse wave velocity. A total of 1272 participants (47% women; mean age: 52+/-13 years; range: 30 to 79 years) from a community-based survey were studied. Carotid pressure waveforms derived by tonometry were decomposed into their forward wave amplitudes, backward wave amplitudes (Pb), and a reflection index (=[Pb/(forward wave amplitude+Pb)]), in addition to augmentation index, Pa, and reflected wave transit time. During a median follow-up of 15 years, 225 deaths occurred (17.6%), including 64 cardiovascular origins (5%). In univariate Cox proportional hazard regression analysis, pulse wave velocity, Pa, and Pb predicted all-cause and cardiovascular mortality in both men and women, whereas augmentation index, reflected wave transit time, and reflection index were predictive only in men. In multivariate analysis accounting for age, height, and heart rate, Pb predicted cardiovascular mortality in both men and women, whereas Pa was predictive only in men. Per 1-SD increment (6 mm Hg), Pb predicted 15-year cardiovascular mortality independent of brachial but not central pressure, pulse wave velocity, augmentation index, Pa, and conventional cardiovascular risk factors with hazard ratios of approximately 1.60 (all P<0.05). In conclusion, Pb, a transit time-independent measure of reflected wave magnitude, predicted long-term cardiovascular mortality in men and women independent of arterial stiffness.

Effect of focal adhesion proteins on endothelial cell adhesion, motility and orientation response to cyclic strain.

Focal adhesion proteins link cell surface integrins and intracellular actin stress fibers and therefore play an important role in mechanotransduction and cell motility. When endothelial cells are subjected to cyclic mechanical strain, time-lapse imaging revealed that cells underwent significant morphological changes with their resultant long axes aligned away from the strain direction. To explore how this response is regulated by focal adhesion-associated proteins the expression levels of paxillin, focal adhesion kinase (FAK), and zyxin were knocked down using gene silencing techniques. In addition, rescue of endogenous and two mutant zyxins were used to investigate the specific role of zyxin interactions. Cells with decreased zyxin expression levels and rescue with the mutant lacking zyxin/alpha-actinin binding exhibited lower orientation angles after comparable times of stretching as compared to normal and control cells. However, knockdown of the expression levels of paxillin and FAK and rescue with the mutant lacking zyxin/VASP (vasodilator-stimulated phosphoprotein) binding did not significantly affect the degree of cell orientation. In addition, wound closure speed and cell-substratum adhesive strength were observed to be significantly reduced only for cells with zyxin depletion and the mutation lacking zyxin/alpha-actinin binding. These results suggest that zyxin and its interaction with alpha-actinin are important in the regulation of endothelial cell adhesive strength, motility and orientation response to mechanical stretching.

A hyperelastic constitutive law for aortic valve tissue.

The objective of the present study was to perform biaxial testing and apply constitutive modeling to develop a strain energy function that accurately predicts the material behavior of the aortic valve leaflets. Ten leaflets from seven normal porcine aortic valves were biaxially stretched in a variety of protocols and the data combined to develop and fit a strain energy function to describe the material behavior. The results showed that the nonlinear anisotropic behavior of the aortic valve is well described by a strain energy function of two strain invariants, which uses only three coefficients to accurately predict the stress-strain behavior over a wide range of deformations. This structurally-motivated constitutive law has many applications, including computational modeling for clinical and engineering valve treatments.

Central or peripheral systolic or pulse pressure: which best relates to target organs and future mortality?

OBJECTIVE: To examine the relationship between brachial and central carotid pressures and target organ indices at baseline and their association with future mortality. METHODS: We examined, cross-sectionally and longitudinally, the relations of baseline systolic and pulse pressures in central (calibrated tonometric carotid pulse) and peripheral (brachial, mercury sphygmomanometer) arteries to baseline left ventricular mass, carotid intima-media thickness, estimated glomerular filtration rate, and 10-year all-cause and cardiovascular mortality in 1272 participants (47% women aged 30-79 years) from a community of homogeneous Chinese. RESULTS: Left ventricular mass was more strongly related to central and peripheral systolic pressures than pulse pressures. Intima-media thickness and glomerular filtration rate were more strongly related to central pressures than peripheral pressures. A total of 130 participants died, 37 from cardiovascular causes. In univariate analysis, all four blood pressure variables significantly predicted all-cause and cardiovascular mortality. Each blood pressure variable was entered into the multivariate models, both individually and jointly with another blood pressure variable. After adjustment for age, sex, heart rate, BMI, current smoking, glucose, ratio of total cholesterol to high-density lipoprotein cholesterol, carotid-femoral pulse wave velocity, left ventricular mass, intima-media thickness, and glomerular filtration rate, only central systolic pressure consistently and independently predicted cardiovascular mortality (hazards ratio, 1.30 per 10 mmHg). No significant sex interactions were observed in all analyses. CONCLUSION: Systolic and pulse pressures relate differently to different target organs. Central systolic pressure is more valuable than other blood pressure variables in predicting cardiovascular mortality.

Mechanical properties of actin stress fibers in living cells.

Actin stress fibers (SFs) play an important role in many cellular functions, including morphological stability, adhesion, and motility. Because of their central role in force transmission, it is important to characterize the mechanical properties of SFs. However, most of the existing studies focus on properties of whole cells or of actin filaments isolated outside cells. In this study, we explored the mechanical properties of individual SFs in living endothelial cells by nanoindentation using an atomic force microscope. Our results demonstrate the pivotal role of SF actomyosin contractile level on mechanical properties. In the same SF, decreasing contractile level with 10 microM blebbistatin decreased stiffness, whereas increasing contractile level with 2 nM calyculin A increased stiffness. Incrementally stretching and indenting SFs made it possible to determine stiffness as a function of strain level and demonstrated that SFs have nearly linear stress-stain properties in the baseline state but nonlinear properties at a lower contractile level. The stiffnesses of peripheral and central portions of the same SF, which were nearly the same in the baseline state, became markedly different after contractile level was increased with calyculin A. Because these results pertain to effects of interventions in the same SF in a living cell, they provide important new understanding about cell mechanics.

Strain-induced orientation response of endothelial cells: effect of substratum adhesiveness and actin-myosin contractile level.

Endothelial cells subjected to cyclic stretching change orientation so as to be aligned perpendicular to the direction of applied strain in a magnitude and time-dependent manner. Although this type of response is not the same as motility, it could be governed by motility-related factors such as substratum adhesiveness and actin-myosin contractile level. To examine this possibility, human aortic endothelial cells (HAEC) were uniaxially, cyclically stretched on silicone rubber membranes coated with various concentrations of fibronectin, collagen type IV and laminin to produce differing amounts of adhesiveness (measured using a radial flow detachment assay). Cells were subjected to 10% pure cyclic uniaxial stretching for three hours at a rate of 10%/sec. Time-lapse images revealed that cells underwent large morphological changes without moving. For each type of protein there was a parabolic dependence on initial adhesiveness with optimal cell orientation occurring at very similar adhesive strengths. The effect of actin-myosin contractile level was examined by stretching cells treated with different doses of 2,3-butanedione monoxime (BDM) and Blebbistatin. Each drug induced a dose-dependent decrease in orientation angles after three hours of cyclic stretching. Furthermore, cell and stress fiber orientations were tightly coupled for untreated and Blebbistatin-treated cells but were uncoupled for BDM-treated cells. Even though orientation response to cyclic stretching is not a spontaneous motile response, it is determined, in large part, by the same factors that affect spontaneous motility--the cell-substratum adhesiveness and actin-myosin contractile level.

Actin stress fiber pre-extension in human aortic endothelial cells.

Actin stress fibers (SFs) enable cells to sense and respond to mechanical stimuli and affect adhesion, motility and apoptosis. We and others have demonstrated that cultured human aortic endothelial cells (HAECs) are internally stressed so that SFs are pre-extended beyond their unloaded lengths. The present study explores factors affecting SF pre-extension. In HAECs cultured overnight the baseline pre-extension was 1.10 and independent of the amount of cell shortening. Decreasing contractility with 30 mM BDM or 10 microM blebbistatin decreased pre-extension to 1.05 whereas increasing contractility with 2 nM calyculin A increased pre-extension to 1.26. Knockdown of alpha-actinin-1 with an interfering RNA increased pre-extension to 1.28. None of these affected the wavelength of the buckled SFs. Pre-extension was the same in unperturbed cells as in those in which the actin cytoskeleton was disrupted by both chemical and mechanical means and then allowed to reassemble. Finally, disrupting MTs or IFs did not affect pre-extension but increased the wavelength. Taken together, these results suggest that pre-extension of SFs is determined primarily by intrinsic factors, i.e. the level of actin-myosin interaction. This intrinsic control of pre-extension is sufficiently robust that pre-extension is the same even after the actin cytoskeleton has been disrupted and reorganized. Unlike pre-extension, the morphology of the compressed SFs is partially determined by MTs and IFs which appear to support the SFs along their lengths.

Pulse pressure is inversely related to aortic root diameter implications for the pathogenesis of systolic hypertension.

Hypertension accelerates the age-associated increase in aortic root diameter (AoD), likely because of chronically elevated distending pressures. However, the pulsatile component of blood pressure may have a different relationship with AoD. We sought to assess the relationship between AoD and pulse pressure (PP) while accounting for left ventricular and central arterial structural and functional properties, which are known to influence PP. The study population was composed of 1256 individuals, aged 30 to 79 years (48% women and 48% hypertensive), none of whom were on antihypertensive medications. Blood pressure was measured in the sitting position with conventional sphygmomanometry. PP was calculated as the difference between systolic and diastolic blood pressures. AoD was measured at end diastole at the level of the sinuses of Valsalva with echocardiography. The relationship between AoD and PP was evaluated with multiple regression analyses. PP was 50+/-14 mm Hg in men and 54+/-18 mm Hg in women, and AoD was 31.9+/-3.5 mm in men and 28.9+/-3.5 mm in women. After adjusting for age, age(2), height, weight, and mean arterial pressure, AoD was independently and inversely associated with PP in both sexes. After further adjustments for central arterial stiffness and wall thickness, reflected waves, and left ventricular geometry, AoD remained inversely associated with PP in both men (coefficient=-0.48; P=0.0003; model R(2)=0.51) and women (coefficient=-0.40; P=0.01; model R(2)=0.61). Thus, AoD is inversely associated with PP, suggesting that a small AoD may contribute to the pathogenesis of systolic hypertension. Longitudinal studies are needed to examine this possibility.

Non-Hertzian approach to analyzing mechanical properties of endothelial cells probed by atomic force microscopy.

Detailed measurements of cell material properties are required for understanding how cells respond to their mechanical environment. Atomic force microscopy (AFM) is an increasingly popular measurement technique that uniquely combines subcellular mechanical testing with high-resolution imaging. However, the standard method of analyzing AFM indentation data is based on a simplified "Hertz" theory that requires unrealistic assumptions about cell indentation experiments. The objective of this study was to utilize an alternative "pointwise modulus" approach, that relaxes several of these assumptions, to examine subcellular mechanics of cultured human aortic endothelial cells (HAECs). Data from indentations in 2- to 5-microm square regions of cytoplasm reveal at least two mechanically distinct populations of cellular material. Indentations colocalized with prominent linear structures in AFM images exhibited depth-dependent variation of the apparent pointwise elastic modulus that was not observed at adjacent locations devoid of such structures. The average pointwise modulus at an arbitrary indentation depth of 200 nm was 5.6+/-3.5 kPa and 1.5+/-0.76 kPa (mean+/-SD, n=7) for these two material populations, respectively. The linear structures in AFM images were identified by fluorescence microscopy as bundles of f-actin, or stress fibers. After treatment with 4 microM cytochalasin B, HAECs behaved like a homogeneous linear elastic material with an apparent modulus of 0.89+/-0.46 kPa. These findings reveal complex mechanical behavior specifically associated with actin stress fibers that is not accurately described using the standard Hertz analysis, and may impact how HAECs interact with their mechanical environment.

Effect of combined cyclic stretch and fluid shear stress on endothelial cell morphological responses.

Endothelial cells in vivo are normally subjected to multiple mechanical stimuli such as stretch and fluid shear stress (FSS) but because each stimulus induces magnitude-dependent morphologic responses, the relative importance of each stimulus in producing the normal in vivo state is not clear Using cultured human aortic endothelial cells, this study first determined equipotent levels of cyclic stretch, steady FSS, and oscillatory FSS with respect to the time course of cell orientation. We then tested whether these levels of stimuli were equipotent in combination with each other by imposing simultaneous cyclic stretch and steady FSS or cyclic stretch and oscillatory FSS so as to reinforce or counteract the cells' orientation responses. Equipotent levels of the three stimuli were 2% cyclic stretch at 2%/s, 80 dynes/cm2 steady FSS and 20 +/- 10 dynes/cm2 oscillatory FSS at 20 dyne/cm2-s. When applied in reinforcing fashion, cyclic stretch and oscillatory, but not steady, FSS were additive. Both pairs of stimuli canceled when applied in counteracting fashion. These results indicate that this level of cyclic stretch and oscillatory FSS sum algebraically so that they are indeed equipotent. In addition, oscillatory FSS is a stronger stimulus than steady FSS for inducing cell orientation. Moreover, arterial endothelial cells in vivo are likely receiving a stronger stretch than FSS stimulus.

Comparison of the effects of cyclic stretching and compression on endothelial cell morphological responses.

Recent results demonstrate the exquisite sensitivity of cell orientation responses to the pattern of imposed deformation. Cells undergoing pure in-plane uniaxial stretching orient differently than cells that are simply elongated--likely because the latter stimulus produces simultaneous compression in the unstretched direction. It is not known, however, if cells respond differently to pure stretching than to pure compression. This study was performed to address this issue. Human aortic endothelial cells were seeded on deformable silicone membranes and subjected to various magnitudes and rates of pure stretching or compression. The cell orientation and cytoskeletal stress fiber organization responses were examined. Both stretching and compression resulted in magnitude-dependent but not rate-dependent orientation responses away from the deforming direction. Compression produced a slower temporal response than stretching. However, stress fiber reorganization responses-early disruption followed by reassembly into parallel arrays along the cells' long axes were similar between the two stimuli. Moreover, the cell orientation and stress fiber responses appeared to be uncoupled since disruption of stress fibers was not required for the cell orientation. Moreover, parallel actin stress fibers were observed at oblique angles to the deforming direction indicating that stress fibers can reassemble when undergoing deformation.

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity.

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC(50))) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching -5.15+/-0.79 and before stretching -6.71+/-0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.

Buckling of actin stress fibers: a new wrinkle in the cytoskeletal tapestry.

Intracellular tension is considered an important determinant of cytoskeletal architecture and cell function. However, many details about cytoskeletal tension remain poorly understood because these forces cannot be directly measured in living cells. Therefore, we have developed a method to characterize the magnitude and distribution of pre-extension of actin stress fibers (SFs) due to resting tension in the cytoskeleton. Using a custom apparatus, human aortic endothelial cells (HAECs) were cultured on a pre-stretched silicone substrate coated with a fibronectin-like polymer. Release of the substrate caused SFs aligned in the shortening direction in adhered cells to buckle when compressed rapidly (5% shortening per second or greater) beyond their unloaded slack length. Subsequently, the actin cytoskeleton completely disassembled in 5 sec and reassembled within 60 sec. Quantification of buckling in digital fluorescent micrographs of cells fixed and stained with rhodamine phalloidin indicated a nonuniform distribution of 0-26% pre-extension of SFs in non-locomoting HAECs. Local variability suggests heterogeneity of cytoskeletal tension and/or stiffness within individual cells. These findings provide new information about the magnitude and distribution of cytoskeletal tension and the dynamics of actin stress fibers, and the approach offers a novel method to elucidate the role of specific cytoskeletal elements and crosslinking proteins in the force generating apparatus of non-muscle cells.