Three-dimensional intracellular optical coherence phase imaging.

Quantitative phase imaging has many applications for label-free studies of the nanoscale structure and dynamics of cells and tissues. It has been demonstrated that optical coherence phase microscopy (OCPM) can provide quantitative phase information with very high sensitivity. The excellent phase stability of OCPM is obtained by use of a reflection from the microscope cover glass as a local reference field. For detailed intracellular studies a large numerical aperture (N.A.) objective is needed in order to obtain the required resolution. Unfortunately, this also means that the depth of field becomes too small to obtain sufficient power from the cover glass when the beam is focused into the sample. To address this issue, we designed a setup with a dual-beam sample arm. One beam with a large diameter (filling the 1.2 N.A. water immersion objective) enabled high-resolution imaging. A second beam with a small diameter (underfilling the same objective) had a larger depth of field and could detect the cover glass used as a local phase reference. The phase stability of the setup was quantified by monitoring the front and back of a cover glass. The standard deviation of the phase difference was 0.021 rad, corresponding to an optical path displacement of 0.9 nm. The lateral and axial dimensions of the confocal point spread function were 0.42 and 0.84 µm, respectively. This makes our dual-beam setup ideal for three-dimensional intracellular phase imaging.

High speed miniature motorized endoscopic probe for optical frequency domain imaging.

We present a miniature motorized endoscopic probe for Optical Coherence Tomography with an outer diameter of 1.65 mm and a rotation speed of 3,000-12,500 rpm. This is the smallest motorized high speed OCT probe to our knowledge. The probe has a motorized distal end which provides a significant advantage over proximally driven probes since it does not require a drive shaft to transfer the rotational torque to the distal end of the probe and functions without a fiber rotary junction. The probe has a focal Full Width at Half Maximum of 9.6 µm and a working distance of 0.47 mm. We analyzed the non uniform rotation distortion and found a location fluctuation of only 1.87° in repeated measurements of the same object. The probe was integrated in a high-speed Optical Frequency Domain Imaging setup at 1310 nm to acquire images from ex vivo pig lung tissue through the working channel of a human bronchoscope.

Early onset of retrograde flow in the main pulmonary artery is a characteristic of pulmonary arterial hypertension.

PURPOSE: To evaluate if early onset of retrograde flow in the main pulmonary artery is a characteristic of pulmonary arterial hypertension (PAH). MATERIALS AND METHODS: Fifty-five patients with suspected pulmonary hypertension (PH) underwent right-sided heart catheterization and retrospectively ECG-gated MR phase-contrast velocity quantification in the main pulmonary artery. Pulmonary hypertension was defined by a mean pulmonary artery pressure being larger than 25 mmHg. The onset time of the retrograde flow relative to the cardiac cycle duration (Relative Onset Time = ROT) was compared with mean pulmonary artery pressure. RESULTS: By the catheterization, 38 patients were identified as having PAH. The ROT for these PAH patients was significantly different from those found in the 17 non-PH subjects (0.14 ± 0.06 versus 0.37 ± 0.06, P < 0.001). The mean pulmonary artery pressure was related to the ROT (r(2) = 0.62, P < 0.001) and could be estimated from the ROT with a standard deviation of 11.7 mmHg. With a cutoff value of 0.25, the ROT distinguished PAH patients from non-PH subjects. CONCLUSION: Early onset of retrograde flow in the main pulmonary artery is a characteristic of pulmonary arterial hypertension and is visible by standard MR phase-contrast velocity quantification.

Right ventricular oscillatory power is a constant fraction of total power irrespective of pulmonary artery pressure.

RATIONALE: Pulmonary hypertension (PH) is characterized by increased arterial load requiring more right ventricular (RV) hydraulic power to sustain adequate forward blood flow. Power can be separated into a mean and oscillatory part. The former is associated with mean and the latter with pulsatile blood flow and pressure. Because mean power provides for net blood flow, the ratio of oscillatory to total power (oscillatory power fraction) preferably should be small. It is unknown whether this is the case in pulmonary arterial hypertension (PAH). OBJECTIVES: To derive components of power generated by the right ventricle in PAH. MEASUREMENTS AND MAIN RESULTS: Thirty-five patients with idiopathic PAH (IPAH) and 14 subjects without PH were included. The patients were divided in two groups, "moderate" and "high," based on pulmonary artery (PA) pressure. PA pressures were obtained by right heart catheterization and PA flows by magnetic resonance imaging. Total hydraulic power (Power(total)) was calculated as the integral product of pressure and flow. Mean hydraulic power (Power(mean)) was calculated as mean pulmonary artery pressure times mean flow. Their difference is oscillatory power (Power(oscill)). Total hydraulic power in subjects without PH compared with moderate and high IPAH was 0.29 ± 0.10 W (n = 14), 0.52 ± 0.14 W (n = 17), and 0.73 ± 0.24 W (n = 18), respectively. The oscillatory power fraction is approximately 23% and not different between groups. CONCLUSIONS: In this study, oscillatory power fraction is constant at 23% in non-PH and IPAH, implying that a considerable amount of power is not used for forward flow, making the RV less efficient with respect to its arterial load. Our findings emphasize the need to develop new therapy strategies to optimize RV power output in PAH.

Progressive dilatation of the main pulmonary artery is a characteristic of pulmonary arterial hypertension and is not related to changes in pressure.

BACKGROUND: Pulmonary artery (PA) dilatation is one of the consequences of pulmonary arterial hypertension (PAH) and is used for noninvasive detection. However, it is unclear how the size of the PA behaves over time and whether it is related to pressure changes. The aim of this study was to evaluate PA size during follow-up in treated patients with PAH and whether it reflects pulmonary vascular hemodynamics. METHODS: Fifty-one patients with PAH who underwent at least two right-sided heart catheterizations (RHCs) together with cardiac MRI (CMR) were included in this study. Another 18 patients who had normal pressure at RHC were included for comparison at baseline. From RHC, we derived PA pressures and cardiac output. From the CMR images we derived PA diameter (PAD) and the ratio of the PAD and ascending aorta diameter. RESULTS: The PAD was significantly larger in patients with PAH than in patients without PAH (P < .001). A ratio of the PAD and ascending aorta diameter > 1 had a positive predictive value of 92% for PAH. Mean follow-up time was 942 days, and there was a significant dilatation during this period (P < .001). The change of the PAD did not correlate with the changes in pressure or cardiac output. A moderate correlation with follow-up time was found (r = 0.56; P < .001). CONCLUSIONS: A dilatated PA is useful for identifying patients with PAH. However, during patient follow-up, progressive dilatation of the PA is independent of the change in PA pressure and cardiac output and might become independent from hemodynamics.

Pulmonary artery size and function after Fontan operation at a young age.

PURPOSE: To assess pulmonary artery (PA) size, flow variables, and wall shear stress (WSS) in patients after Fontan operation at a young age. MATERIALS AND METHODS: Flow in the branch PA was obtained with phase contrast velocity-encoded cardiovascular magnetic resonance imaging in 14 patients before and after low-dose dobutamine stress (7.5 microg/kg/min) and in 17 healthy controls at rest. RESULTS: At rest, stroke index, total flow, average, and peak flow rate were all statistically significantly lower in patients than in controls (P<0.001). With stress-testing, all variables increased in patients (P<0.001), apart from stroke index, which did not change. At rest, branch PA area did not differ between patients and controls. Distensibility was lower in patients than in controls (P<0.001). With stress-testing, area and distensibility did not change. At rest, WSS was lower in patients than in controls (P<0.001). WSS increased with stress-testing (P<0.001), but not to the same levels as during resting conditions of the control group. CONCLUSION: PA size is normal long-term after Fontan operation at a young age. Flow variables, distensibility, and WSS are significantly lower compared to healthy controls, and do not show adequate reactions with stress-testing, which is suggestive of pulmonary artery endothelial and/or vascular dysfunction.

Rapamycin modulates the eNOS vs. shear stress relationship.

AIMS: Studies in animals and patients indicate that rapamycin affects vasodilatation differently in outer and inner curvatures of blood vessels. We evaluated in this study whether rapamycin affects endothelial nitric oxide synthase (eNOS) responsiveness to shear stress under normo- and hypercholesteraemic conditions to explain these findings. METHODS AND RESULTS: Shear stress levels were varied over a large range of values in carotid arteries of transgenic mice expressing human eNOS fused to enhanced green fluorescence protein. The mice were divided into control, low-dose rapamycin (3 microg/kg/day), and high-dose rapamycin (3 mg/kg/day) groups and into normocholesteraemic and hypercholesteraemic (ApoE-/- on high cholesterol diet for 3-4 weeks) groups. The effect of rapamycin treatment on eNOS was evaluated by quantification of eNOS expression and of intracellular protein levels by en face confocal microscopy. A sigmoid curve fit was used to described these data. The efficacy of treatment was confirmed by measurement of rapamycin serum levels (2.0 +/- 0.5 ng/mL), and of p27kip1 expression in vascular tissue (increased by 2.4 +/- 0.5-fold). In control carotid arteries, eNOS expression increased by 1.8 +/- 0.3-fold in response to rapamycin. In the treated vessels, rapamycin reduced maximal eNOS expression at high shear stress levels (>5 Pa) in a dose-dependent way and shifted the sigmoid curve to the right. Hypercholesteraemia had a tendency to increase the leftward shift and the reduction in maximal eNOS expression (P = 0.07). CONCLUSION: Rapamycin is associated with high eNOS in low shear regions, i.e. in atherogenic regions, protecting these regions against atherosclerosis, and is associated with a reduction of eNOS at high shear stress affecting vasomotion in these regions.

Effect of shear stress on vascular inflammation and plaque development.

PURPOSE OF REVIEW: This review describes evidence that shear stress acts through modulation of inflammation and by that process affects atherogenesis and plaque composition. RECENT FINDINGS: In low shear stress regions antiatherogenic transcription factors are downregulated and pro-atherogenic transcription factors are upregulated. Consequently, inflammatory cells may home low shear stress regions more easily to the plaques because of increased expression of adhesion factors, a decreased rolling speed and an increased expression of chemokines, thereby changing the composition of the plaques into a more vulnerable phenotype. In contrast, in advanced plaque development vascular lumen decreases and shear stress increases, especially upstream of the plaques. The predominant upstream location of lipids induces a prevalent upstream location of inflammatory cells leading to localized plaque rupture. SUMMARY: Shear stress has been shown to play a role in plaque induction, plaque progression and plaque rupture. The mechanism for plaque induction seems to differ from the role of shear stress for plaque rupture, whereby the former mechanism is induced by low shear stress and the latter by high shear stress.

Gelatinolytic activity in atherosclerotic plaques is highly localized and is associated with both macrophages and smooth muscle cells in vivo.

BACKGROUND: Atherosclerosis is considered an inflammatory disease. Recent studies provided evidence for a predominant upstream location of plaque inflammation. The present study introduces a novel technique that evaluates the underlying mechanism of this spatial organization. METHODS AND RESULTS: In hypercholesterolemic rabbits, atherosclerosis of the infrarenal aorta was induced by a combination of endothelial denudation and a high-cholesterol diet (2% cholesterol for 2 months). At the time of death, aortic vessel segments were dissected and reconstructed with a new technique that preserved the original intravascular ultrasound-derived lumen geometry. This enabled us to study the spatial relation of histological markers like macrophages, smooth muscle cells, lipids, gelatinolytic activity, and oxidized low-density lipoprotein. Results showed a predominant upstream localization of macrophages and gelatinase activity. Colocalization studies indicated that gelatinase activity was associated with macrophages and smooth muscle cells. Further analysis revealed that this was caused by subsets of smooth muscle cells and macrophages, which were associated with oxidized low-density lipoprotein accumulation. CONCLUSIONS: Upstream localization of a vulnerable plaque phenotype is probably due to an accumulation of oxidized low-density lipoprotein, which activates/induces subsets of smooth muscle cells and macrophages to gelatinase production.

Large variations in absolute wall shear stress levels within one species and between species.

Wall shear stress (WSS), the frictional force between blood and endothelium, is an important determinant of vascular function. It is generally assumed that WSS remains constant at a reference value of 15 dyn/cm(2). In a study of small rodents, we realized that this assumption could not be valid. This review presents an overview of recent studies in large and small animals where shear stress was measured, derived from velocity measurements or otherwise, in large vessels. The data show that large variations exist within a single species (human: variation of 2-16 N/m(2)). Moreover, when we compared different species at the same location within the arterial tree, an inverse relationship between animal size and wall shear stress was noted. When we related WSS to diameter, a unique relationship was derived for all species studied. This relationship could not be described by the well-known r(3) law of Murray, but by the r(2) law introduced by Zamir et al. in 1972. In summary, by comparing data from the literature, we have shown that: (i) the assumption of a physiological WSS level of approximately 15 dyn/cm(2) for all straight vessels in the arterial tree is incorrect; (ii) WSS is not constant throughout the vascular tree; (iii) WSS varies between species; (iv) WSS is inversely related to the vessel diameter. These data support an "r(2) law" rather than Murray's r(3) law for the larger vessels in the arterial tree.

The role of shear stress in atherosclerosis: action through gene expression and inflammation?

Atherosclerotic lesions preferentially localize near side branches or curved vessels. During the last few decades, research has been shown that low or low and oscillating shear stress is associated with plaque location. Despite ample evidence, the precise mechanism is unknown. This is mainly because of a lack of appropriate animal models. We describe two novel methods to study the hypothesis that shear stress acts through endothelial gene expression or shear stress acts through localizing of inflammation. Both literature evidence and own findings support a role for both mechanisms in atherosclerosis.