Customisable Silicone Vessels and Tissue Phantoms for In Vitro Photoplethysmography Investigations into Cardiovascular Disease.

Age-related vessel deterioration leads to changes in the structure and function of the heart and blood vessels, notably stiffening of vessel walls, increasing the risk of developing cardiovascular disease (CVD), which accounts for 17.9 million global deaths annually. This study describes the fabrication of custom-made silicon vessels with varying mechanical properties (arterial stiffness). The primary objective of this study was to explore how changes in silicone formulations influenced vessel properties and their correlation with features extracted from signals obtained from photoplethysmography (PPG) reflectance sensors in an in vitro setting. Through alterations in the silicone formulations, it was found that it is possible to create elastomers exhibiting an elasticity range of 0.2 MPa to 1.22 MPa. It was observed that altering vessel elasticity significantly impacted PPG signal morphology, particularly reducing amplitude with increasing vessel stiffness (p < 0.001). A p-value of 5.176 × 10-15 and 1.831 × 10-14 was reported in the red and infrared signals, respectively. It has been concluded in this study that a femoral artery can be recreated using the silicone material, with the addition of a softener to achieve the required mechanical properties. This research lays the foundation for future studies to replicate healthy and unhealthy vascular systems. Additional pathologies can be introduced by carefully adjusting the elastomer materials or incorporating geometrical features consistent with various CVDs.

Arterial stiffness assessment using PPG feature extraction and significance testing in an in vitro cardiovascular system.

Cardiovascular diseases (CVDs) remain the leading cause of global mortality, therefore understanding arterial stiffness is essential to developing innovative technologies to detect, monitor and treat them. The ubiquitous spread of photoplethysmography (PPG), a completely non-invasive blood-volume sensing technology suitable for all ages, highlights immense potential for arterial stiffness assessment in the wider healthcare setting outside specialist clinics, for example during routine visits to a General Practitioner or even at home with the use of mobile and wearable health devices. This study employs a custom-manufactured in vitro cardiovascular system with vessels of varying stiffness to test the hypothesis that PPG signals may be used to detect and assess the level of arterial stiffness under controlled conditions. Analysis of various morphological features demonstrated significant (p < 0.05) correlations with vessel stiffness. Particularly, area related features were closely linked to stiffness in red PPG signals, while for infrared PPG signals the most correlated features were related to pulse-width. This study demonstrates the utility of custom vessels and in vitro investigations to work towards non-invasive cardiovascular assessment using PPG, a valuable tool with applications in clinical healthcare, wearable health devices and beyond.

Photoplethysmography for the Assessment of Arterial Stiffness.

This review outlines the latest methods and innovations for assessing arterial stiffness, along with their respective advantages and disadvantages. Furthermore, we present compelling evidence indicating a recent growth in research focused on assessing arterial stiffness using photoplethysmography (PPG) and propose PPG as a potential tool for assessing vascular ageing in the future. Blood vessels deteriorate with age, losing elasticity and forming deposits. This raises the likelihood of developing cardiovascular disease (CVD), widely reported as the global leading cause of death. The ageing process induces structural modifications in the vascular system, such as increased arterial stiffness, which can cause various volumetric, mechanical, and haemodynamic alterations. Numerous techniques have been investigated to assess arterial stiffness, some of which are currently used in commercial medical devices and some, such as PPG, of which still remain in the research space.

Effects of using different algorithms and fiducial points for the detection of interbeat intervals, and different sampling rates on the assessment of pulse rate variability from photoplethysmography.

OBJECTIVE: Pulse Rate Variability (PRV) has been widely used as a surrogate of Heart Rate Variability (HRV). However, there are several technical aspects that may affect the extraction of PRV information from pulse wave signals such as the photoplethysmogram (PPG). The aim of this study was to evaluate the effects of changing the algorithm and fiducial points used for determining inter-beat intervals (IBIs), as well as the PPG sampling rate, from simulated PPG signals with known PRV content. METHODS: PPG signals were simulated using a proposed model, in which PRV information can be modelled. Two independent experiments were performed. First, 5 IBIs detection algorithms and 8 fiducial points were used for assessing PRV information from the simulated PPG signals, and time-domain and Poincaré plot indices were extracted and compared to the expected values according to the simulated PRV. The best combination of algorithms and fiducial points were determined for each index, using factorial designs. Then, using one of the best combinations, PPG signals were simulated with varying sampling rates. PRV indices were extracted and compared to the expected values using Student t-tests or Mann-Whitney U-tests. RESULTS: From the first experiment, it was observed that AVNN and SD2 indices behaved similarly, and there was no significant influence of the fiducial points used. For other indices, there were several combinations that behaved similarly well, mostly based on the detection of the valleys of the PPG signal. There were differences according to the quality of the PPG signal. From the second experiment, it was observed that, for all indices but SDNN, the higher the sampling rate the better. AVNN and SD2 showed no statistical differences even at the lowest evaluated sampling rate (32 Hz), while RMSSD, pNN50, S, SD1 and SD1/SD2 showed good performance at sampling rates as low as 128 Hz. CONCLUSION: The best combination of IBIs detection algorithms and fiducial points differs according to the application, but those based on the detection of the valleys of the PPG signal tend to show a better performance. The sampling rate of PPG signals for PRV analysis could be lowered to around 128 Hz, although it could be further lowered according to the application. SIGNIFICANCE: The standardisation of PRV analysis could increase the reliability of this signal and allow for the comparison of results obtained from different studies. The obtained results allow for a first approach to establish guidelines for two important aspects in PRV analysis from PPG signals, i.e. the way the IBIs are segmented from PPG signals, and the sampling rate that should be used for these analyses. Moreover, a model for simulating PPG signals with PRV information has been proposed, which allows for the establishing of these guidelines while controlling for other variables, such as the quality of the PPG signal.

Effect of Filtering of Photoplethysmography Signals in Pulse Rate Variability Analysis.

Due to the widespread use and simplicity of photoplethysmography (PPG) signals, and because this signal contains information related to pulse rate, several studies have started to propose the use of Pulse Rate Variability (PRV) for the assessment of cardiovascular autonomic nervous activity, instead of using Heart Rate Variability (HRV) obtained with the electrocardiogram (ECG). However, there is a lack of standardisation and guidelines for the measurement of PRV from PPG signals, which might hinder comparability among studies and validation of results. The aim of this study was to evaluate different digital filters on PPG signals and their effects on PRV information, compared to HRV obtained from ECG. PPG and ECG signals obtained from healthy volunteers were used to measure HRV and PRV. PPG signals were filtered using different FIR and IIR digital filters, with several cut-off frequencies. The results indicate that filtering PPG signals using IIR filters and lower low-cut-off frequencies allow for the acquisition of more reliable PRV information, with lower Bland-Altman ratios and higher cross-correlations when compared to HRV. This is a first step in establishing guidelines and standards for the analysis of PRV information using PPG signals.Clinical relevance- Pulse rate variability might be a useful tool for the assessment of the cardiovascular autonomic nervous system. This study is the first step for establishing standards of measurement of this signal, which helps in the comparability and validation of the technique.

In-Vitro Investigation of Flow Profiles in Arteries Using the Photoplethysmograph.

The flow profile in the artery reflects the health status of the vessel and generally the arterial system. The aim of this pilot study was to investigate in-vitro the effect of flow profiles on reflective photoplethysmography (PPG) signals at different steady state flow rates and levels of vessel constrictions. A simplified model of an arterial system was built, consisting of a steady state flow gear pump, PVC vinyl tubing, reservoir and a clamp with a micrometer gauge. The blood mimicking fluid (2.5% India ink and water solution) was pumped through the model. It was found that the waveforms of the PPG signals fluctuate irregularly and the magnitude of the frequency components was increased below 60 Hz in cases of turbulent flow (Re = 2503). These preliminary results suggest that PPG could be the basis for new technologies for assessing the profile of the blood flow in the artery. Future studies have to be carried out with pulsatile flow and more complex models that are more similar to the human arterial system.Clinical Relevance- The PPG signal reflects changes in the flow profile caused by the stenotic rigid vessel.

Effects of Contact Pressure in Reflectance Photoplethysmography in an In Vitro Tissue-Vessel Phantom.

With the continued development and rapid growth of wearable technologies, PPG has become increasingly common in everyday consumer devices such as smartphones and watches. There is, however, minimal knowledge on the effect of the contact pressure exerted by the sensor device on the PPG signal and how it might affect its morphology and the parameters being calculated. This study explores a controlled in vitro study to investigate the effect of continually applied contact pressure on PPG signals (signal-to-noise ratio (SNR) and 17 morphological PPG features) from an artificial tissue-vessel phantom across a range of simulated blood pressure values. This experiment confirmed that for reflectance PPG signal measurements for a given anatomical model, there exists an optimum sensor contact pressure (between 35.1 mmHg and 48.1 mmHg). Statistical analysis shows that temporal morphological features are less affected by contact pressure, lending credit to the hypothesis that for some physiological parameters, such as heart rate and respiration rate, the contact pressure of the sensor is of little significance, whereas the amplitude and geometric features can show significant change, and care must be taken when using morphological analysis for parameters such as SpO2 and assessing autonomic responses.

Classification of blood pressure in critically ill patients using photoplethysmography and machine learning.

OBJECTIVE: The aim of this study was to evaluate the capability of features extracted from photoplethysmography (PPG) based Pulse Rate Variability (PRV) to classify hypertensive, normotensive and hypotensive events, and to estimate mean arterial, systolic and diastolic blood pressure in critically ill patients. METHODS: Time-domain, frequency-domain and non-linear indices from PRV were extracted from 5-min and 1-min segments obtained from PPG signals. These features were filtered using machine learning algorithms in order to obtain the optimal combination for the classification of hypertensive, hypotensive and normotensive events, and for the estimation of blood pressure. RESULTS: 5-min segments allowed for an improved performance in both classification and estimation tasks. Classification of blood pressure states showed around 70% accuracy and around 75% specificity. The sensitivity, precision and F1 scores were around 50%. In estimating mean arterial, systolic, and diastolic blood pressure, mean absolute errors as low as 2.55 ± 0.78 mmHg, 4.74 ± 2.33 mmHg, and 1.78 ± 0.14 mmHg were obtained, respectively. Bland-Altman analysis and Wilcoxon rank sum tests showed good agreement between real and estimated values, especially for mean and diastolic arterial blood pressures. CONCLUSION: PRV-based features could be used for the classification of blood pressure states and the estimation of blood pressure values, although including additional features from the PPG waveform could improve the results. SIGNIFICANCE: PRV contains information related to blood pressure, which may aid in the continuous, noninvasive, non-intrusive estimation of blood pressure and detection of hypertensive and hypotensive events in critically ill subjects.

Differential effects of the blood pressure state on pulse rate variability and heart rate variability in critically ill patients.

Heart rate variability (HRV) utilizes the electrocardiogram (ECG) and has been widely studied as a non-invasive indicator of cardiac autonomic activity. Pulse rate variability (PRV) utilizes photoplethysmography (PPG) and recently has been used as a surrogate for HRV. Several studies have found that PRV is not entirely valid as an estimation of HRV and that several physiological factors, including the pulse transit time (PTT) and blood pressure (BP) changes, may affect PRV differently than HRV. This study aimed to assess the relationship between PRV and HRV under different BP states: hypotension, normotension, and hypertension. Using the MIMIC III database, 5 min segments of PPG and ECG signals were used to extract PRV and HRV, respectively. Several time-domain, frequency-domain, and nonlinear indices were obtained from these signals. Bland-Altman analysis, correlation analysis, and Friedman rank sum tests were used to compare HRV and PRV in each state, and PRV and HRV indices were compared among BP states using Kruskal-Wallis tests. The findings indicated that there were differences between PRV and HRV, especially in short-term and nonlinear indices, and although PRV and HRV were altered in a similar manner when there was a change in BP, PRV seemed to be more sensitive to these changes.

Heart Rate Variability and Multi-Site Pulse Rate Variability for the Assessment of Autonomic Responses to Whole-Body Cold Exposure.

Heart rate variability (HRV) is a noninvasive marker of cardiac autonomic activity and has been used in different circumstances to assess the autonomic responses of the body. Pulse rate variability (PRV), a similar variable obtained from pulse waves, has been used in recent years as a valid surrogate of HRV. However, the effect that localized changes in autonomic activity have in the relationship between HRV and PRV has not been entirely understood. In this study, a whole-body cold exposure protocol was performed to generate localized changes in autonomic activity, and HRV and PRV from different body sites were obtained. PRV measured from the earlobe and the finger was shown to differ from HRV, and the correlation between these variables was affected by the cold. Also, it was found that PRV from the finger was more affected by cold exposure than PRV from the earlobe. In conclusion, PRV is affected differently to HRV when localized changes in autonomic activity occur. Hence, PRV should not be considered as a valid surrogate of HRV under certain circumstances.Clinical Relevance- This indicates that pulse rate variability is affected differently to heart rate variability when autonomic activity is modified and suggests that pulse rate variability is not always a valid surrogate of heart rate variability.

Fabricating Novel PDMS Vessels for Phantoms in Photoplethysmography Investigations.

This paper introduces a novel technique for the development of custom polydimethylsiloxane (PDMS) vessels for use in phantom technologies. The method involves continuous dip coating of commercial silicone tubes with rapid curation in a single controlled process. The technique accommodates the production of different vessel diameters, wall thicknesses (56 µm-80 µm) and mechanical properties. Clear phantoms were fabricated to compare the commercial silicone tubes against the customs vessels. A pulsatile fluidic pump (BDCLabs, CO, USA) driven by a computer controlled linear motor generated the pulsatile flow through the phantom. The resulting flow profile, using the custom vessels, simulates human blood flow and the detected contact PPG signal from the phantom closely resembles the morphology of in vivo PPG waveforms with signal-to-noise ratios of 38.16 dB and 40.59 dB, compared to the closest commercially-available tubing at 5.38 dB and 10.59 dB for the red and infrared wavelengths respectively. The rigidity and thick walls of commercial silicone tubes impede the expansion of the tubing under systolic pressure. This technique eliminates this common limitation in phantom development.

Heart Rate Variability (HRV) and Pulse Rate Variability (PRV) for the Assessment of Autonomic Responses.

Introduction: Heart Rate Variability (HRV) and Pulse Rate Variability (PRV), are non-invasive techniques for monitoring changes in the cardiac cycle. Both techniques have been used for assessing the autonomic activity. Although highly correlated in healthy subjects, differences in HRV and PRV have been observed under various physiological conditions. The reasons for their disparities in assessing the degree of autonomic activity remains unknown. Methods: To investigate the differences between HRV and PRV, a whole-body cold exposure (CE) study was conducted on 20 healthy volunteers (11 male and 9 female, 30.3 ± 10.4 years old), where PRV indices were measured from red photoplethysmography signals acquired from central (ear canal, ear lobe) and peripheral sites (finger and toe), and HRV indices from the ECG signal. PRV and HRV indices were used to assess the effects of CE upon the autonomic control in peripheral and core vasculature, and on the relationship between HRV and PRV. The hypotheses underlying the experiment were that PRV from central vasculature is less affected by CE than PRV from the peripheries, and that PRV from peripheral and central vasculature differ with HRV to a different extent, especially during CE. Results: Most of the PRV time-domain and Poincaré plot indices increased during cold exposure. Frequency-domain parameters also showed differences except for relative-power frequency-domain parameters, which remained unchanged. HRV-derived parameters showed a similar behavior but were less affected than PRV. When PRV and HRV parameters were compared, time-domain, absolute-power frequency-domain, and non-linear indices showed differences among stages from most of the locations. Bland-Altman analysis showed that the relationship between HRV and PRV was affected by CE, and that it recovered faster in the core vasculature after CE. Conclusion: PRV responds to cold exposure differently to HRV, especially in peripheral sites such as the finger and the toe, and may have different information not available in HRV due to its non-localized nature. Hence, multi-site PRV shows promise for assessing the autonomic activity on different body locations and under different circumstances, which could allow for further understanding of the localized responses of the autonomic nervous system.

Novel Polydimethylsiloxane (PDMS) Pulsatile Vascular Tissue Phantoms for the In-Vitro Investigation of Light Tissue Interaction in Photoplethysmography.

Currently there exists little knowledge or work in phantoms for the in-vitro evaluation of photoplethysmography (PPG), and its' relationship with vascular mechanics. Such phantoms are needed to provide robust, basic scientific knowledge, which will underpin the current efforts in developing new PPG technologies for measuring or estimating blood pressure, blood flow and arterial stiffness, to name but a few. This work describes the design, fabrication and evaluation of finger tissue-simulating pulsatile phantoms with integrated custom vessels. A novel technique has been developed to produce custom polydimethylsiloxane (PDMS) vessels by a continuous dip-coating process. This process can accommodate the production of different sized vessel diameters (1400-2500 µm) and wall thicknesses (56-80 µm). These vessels were embedded into a mould with a solution of PDMS and India ink surrounding them. A pulsatile pump experimental rig was set up to test the phantoms, where flow rate (1-12 L·min-1), heart rate (40-120 bpm), and total resistance (0-100% resistance clamps) could be controlled on demand. The resulting flow profiles approximates human blood flow, and the detected contact PPG signal (red and infrared) from the phantom closely resembles the morphology of in-vivo PPG waveforms with signal-to-noise ratios of 38.16 and 40.59 dB, for the red and infrared wavelengths, respectively. The progress made by this phantom development will help in obtaining new knowledge in the behaviour of PPG's under differing flow conditions, optical tissue properties and differing vessel stiffness.

Pulse rate variability in cardiovascular health: a review on its applications and relationship with heart rate variability.

Heart rate variability has been largely used for the assessment of cardiac autonomic activity, due to the direct relationship between cardiac rhythm and the activity of the sympathetic and parasympathetic nervous system. In recent years, another technique, pulse rate variability, has been used for assessing heart rate variability information from pulse wave signals, especially from photoplethysmography, a non-invasive, non-intrusive, optical technique that measures the blood volume in tissue. The relationship, however, between pulse rate variability and heart rate variability is not entirely understood, and the effects of cardiovascular changes in pulse rate variability have not been thoroughly elucidated. In this review, a comprehensive summary of the applications in which pulse rate variability has been used, with a special focus on cardiovascular health, and of the studies that have compared heart rate variability and pulse rate variability is presented. It was found that the relationship between heart rate variability and pulse rate variability is not entirely understood yet, and that pulse rate variability might be influenced not only due to technical aspects but also by physiological factors that might affect the measurements obtained from pulse-to-pulse time series extracted from pulse waves. Hence, pulse rate variability must not be considered as a valid surrogate of heart rate variability in all scenarios, and care must be taken when using pulse rate variability instead of heart rate variability. Specifically, the way pulse rate variability is affected by cardiovascular changes does not necessarily reflect the same information as heart rate variability, and might contain further valuable information. More research regarding the relationship between cardiovascular changes and pulse rate variability should be performed to evaluate if pulse rate variability might be useful for the assessment of not only cardiac autonomic activity but also for the analysis of mechanical and vascular autonomic responses to these changes.

A Novel Photoplethysmography Sensor for Vital Signs Monitoring from the Human Trachea.

Current pulse oximeter sensors can be challenged in working accurately and continuously in situations of reduced periphery perfusion, especially among anaesthetised patients. A novel tracheal photoplethysmography (PPG) sensor has been developed in an effort to address the limitations of current pulse oximeters. The sensor has been designed to estimate oxygen saturation (SpO2) and pulse rate, and has been manufactured on a flexible printed circuit board (PCB) that can adhere to a standard endotracheal (ET) tube. A pilot clinical trial was carried out as a feasibility study on 10 anaesthetised patients. Good quality PPGs from the trachea were acquired at red and infrared wavelengths in all patients. The mean SpO2 reading for the ET tube was 97.1% (SD 1.0%) vs. the clinical monitor at 98.7% (SD 0.7%). The mean pulse rate for the ET sensor was 65.4 bpm (SD 10.0 bpm) vs. the clinical monitor at 64.7 bpm (SD 9.9 bpm). This study supports the hypothesis that the human trachea could be a suitable monitoring site of SpO2 and other physiological parameters, at times where the periphery circulation might be compromised.

A Pulsatile Optical Tissue Phantom for the Investigation of Light-Tissue Interaction in Reflectance Photoplethysmography.

The aim of this study was to investigate the effect of emitter-detector separation distance and arterial depth in reflectance photoplethysmography (PPG), utilizing a homogenous pulsatile phantom that exhibits the broad optical absorbance and scattering properties of human tissue. The developed phantom comprised of embedded silicone arteries (outer diameter = 4 mm) that were arranged parallel to one another at nine increasing depths (3.2 mm to 24.4 mm). A pulsatile pump (Harvard Apparatus, MA, USA) circulated a blood imitating fluid through the vessels at the desired heart rate (60 bpm) and stroke volume (5 Lmin-1). The PPG sensor's emitter and detector were isolated on a translation bridge to provide a computer-controlled separation distance between them. Recordings were taken at each vessel depth for emitter-detector separation distances from 2 mm to 8 mm in 0.1 mm steps. The optimum separation distance between the emitter and detector for vessels between depths of 3.2 mm and 10.5 mm was between 3.7 and 4.3 mm, suggesting that the maximum penetration of IR (930 nm) light in a homogenous pulsatile phantom is no greater than 10.5 mm.

Loss of 2 Akt (Protein Kinase B) Isoforms in Hematopoietic Cells Diminished Monocyte and Macrophage Survival and Reduces Atherosclerosis in Ldl Receptor-Null Mice.

Objective- Macrophages express 3 Akt (protein kinase B) isoforms, Akt1, Akt2, and Akt3, which display isoform-specific functions but may be redundant in terms of Akt survival signaling. We hypothesize that loss of 2 Akt isoforms in macrophages will suppress their ability to survive and modulate the development of atherosclerosis. Approach and Results- To test this hypothesis, we reconstituted male Ldlr-/- mice with double Akt2/Akt3 knockout hematopoietic cells expressing only the Akt1 isoform (Akt1only). There were no differences in body weight and plasma lipid levels between the groups after 8 weeks of the Western diet; however, Akt1only→ Ldlr-/- mice developed smaller (57.6% reduction) atherosclerotic lesions with more apoptotic macrophages than control mice transplanted with WT (wild type) cells. Next, male and female Ldlr-/- mice were reconstituted with double Akt1/Akt2 knockout hematopoietic cells expressing the Akt3 isoform (Akt3only). Female and male Akt3only→ Ldlr-/- recipients had significantly smaller (61% and 41%, respectively) lesions than the control WT→ Ldlr-/- mice. Loss of 2 Akt isoforms in hematopoietic cells resulted in markedly diminished levels of white blood cells, B cells, and monocytes and compromised viability of monocytes and peritoneal macrophages compared with WT cells. In response to lipopolysaccharides, macrophages with a single Akt isoform expressed low levels of inflammatory cytokines; however, Akt1only macrophages were distinct in expressing high levels of antiapoptotic Il10 compared with WT and Akt3only cells. Conclusions- Loss of 2 Akt isoforms in hematopoietic cells, preserving only a single Akt1 or Akt3 isoform, markedly compromises monocyte and macrophage viability and diminishes early atherosclerosis in Ldlr-/- mice.

Effect of Impact Mechanism on Head Accelerations in Men's Lacrosse Athletes.

Quantifying head impacts is a vital component to understanding and preventing head trauma in sport. Our objective was to establish the frequency and magnitude of head impact mechanisms in men's lacrosse athletes. Eleven male lacrosse athletes wore xPatch sensors during activity. Video footage of practices and games was analyzed to verify impacts and code them with impact mechanisms. The authors calculated incidence rates (IRs) per 1000 exposures with corresponding 95% confidence intervals (CIs) and used multivariate analysis of variances to compare the linear (g) and rotational (rad/s2) accelerations between mechanisms. A total of 167 head impacts were successfully verified and coded with a mechanism using video footage during 542 total exposures. The highest IR was head to body (IR = 118.08; 95% CI, 89.15-147.01), and the lowest was head to ball (IR = 3.69; 95% CI, 0-8.80) (incidence rate ratio = 32.00; 95% CI, 67.83-130.73). Analysis indicated that impact mechanism failed to significantly alter the combined dependent variables (multivariate F10,306 = 1.79, P = .06, η2 = .06, 1-ß = 0.83). While head to head, body to head, and stick to head mechanisms are penalty-inducing offenses in men's lacrosse, head to ground, head to ball, and combination impacts have similar head accelerations. If penalties and rules are created to protect players from traumatic head injury, the authors recommend stricter enforcement.

Loss of Rictor in Monocyte/Macrophages Suppresses Their Proliferation and Viability Reducing Atherosclerosis in LDLR Null Mice.

Background: Rictor is an essential component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2), a conserved serine/threonine kinase that may play a role in cell proliferation, survival and innate or adaptive immune responses. Genetic loss of Rictor inactivates mTORC2, which directly activates Akt S473 phosphorylation and promotes pro-survival cell signaling and proliferation. Methods and results: To study the role of mTORC2 signaling in monocytes and macrophages, we generated mice with myeloid lineage-specific Rictor deletion (MRictor-/-). These MRictor-/- mice exhibited dramatic reductions of white blood cells, B-cells, T-cells, and monocytes but had similar levels of neutrophils compared to control Rictor flox-flox (Rictorfl/fl) mice. MRictor-/- bone marrow monocytes and peritoneal macrophages expressed reduced levels of mTORC2 signaling and decreased Akt S473 phosphorylation, and they displayed significantly less proliferation than control Rictorfl/fl cells. In addition, blood monocytes and peritoneal macrophages isolated from MRictor-/- mice were significantly more sensitive to pro-apoptotic stimuli. In response to LPS, MRictor-/- macrophages exhibited the M1 phenotype with higher levels of pro-inflammatory gene expression and lower levels of Il10 gene expression than control Rictorfl/fl cells. Further suppression of LPS-stimulated Akt signaling with a low dose of an Akt inhibitor, increased inflammatory gene expression in macrophages, but genetic inactivation of Raptor reversed this rise, indicating that mTORC1 mediates this increase of inflammatory gene expression. Next, to elucidate whether mTORC2 has an impact on atherosclerosis in vivo, female and male Ldlr null mice were reconstituted with bone marrow from MRictor-/- or Rictorfl/fl mice. After 10 weeks of the Western diet, there were no differences between the recipients of the same gender in body weight, blood glucose or plasma lipid levels. However, both female and male MRictor-/- → Ldlr-/- mice developed smaller atherosclerotic lesions in the distal and proximal aorta. These lesions contained less macrophage area and more apoptosis than lesions of control Rictorfl/fl → Ldlr-/- mice. Thus, loss of Rictor and, consequently, mTORC2 significantly compromised monocyte/macrophage survival, and this markedly diminished early atherosclerosis in Ldlr-/- mice. Conclusion: Our results demonstrate that mTORC2 is a key signaling regulator of macrophage survival and its depletion suppresses early atherosclerosis.

Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin.

BACKGROUND: Increased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity. METHODS: Human umbilical vein endothelial cells (HUVEC) were grown to confluence and treated with CFH with or without ascorbate. Monolayer permeability was measured by Electric Cell-substrate Impedance Sensing (ECIS) or transfer of 14C-inulin. Viability was measured by trypan blue exclusion. Intracellular ascorbate was measured by HPLC. RESULTS: CFH increased permeability in a dose- and time-dependent manner with 1 mg/ml of CFH increasing inulin transfer by 50% without affecting cell viability. CFH (1 mg/ml) also caused a dramatic reduction in intracellular ascorbate in the same time frame (1.4 mM without CFH, 0.23 mM 18 h after 1 mg/ml CFH, p < 0.05). Pre-treatment of HUVECs with ascorbate attenuated CFH induced permeability. CONCLUSIONS: CFH increases endothelial permeability in part through depletion of intracellular ascorbate. Supplementation of ascorbate can attenuate increases in permeability mediated by CFH suggesting a possible therapeutic approach in sepsis.