Proteomic Profiling of Early Secreted Proteins in Response to Lipopolysaccharide-Induced Vascular Endothelial Cell EA.hy926 Injury.

Sepsis is a crucial public health problem with a high mortality rate caused by a dysregulated host immune response to infection. Vascular endothelial cell injury is an important hallmark of sepsis, which leads to multiple organ failure and death. Early biomarkers to diagnose sepsis may provide early intervention and reduce risk of death. Damage-associated molecular patterns (DAMPs) are host nuclear or cytoplasmic molecules released from cells following tissue damage. We postulated that DAMPs could potentially be a novel sepsis biomarker. We used an in vitro model to determine suitable protein-DAMPs biomarkers for early sepsis diagnosis. Low and high lipopolysaccharide (LPS) doses were used to stimulate the human umbilical vein endothelial cell line EA.hy926 for 24, 48, and 72 h. Results showed that cell viability was reduced in both dose-dependent and time-dependent manners. Cell injury was corroborated by a significant increase in lactate dehydrogenase (LDH) activity within 24 h in cell-conditioned medium. Secreted protein-DAMPs in the supernatant, collected at different time points within 24 h, were characterized using shotgun proteomics LC-MS/MS analysis. Results showed that there were 2233 proteins. Among these, 181 proteins from the LPS-stimulated EA.hy926 at 1, 12, and 24 h were significantly different from those of the control. Twelve proteins were up-regulated at all three time points. Furthermore, a potential interaction analysis of predominant DAMPs-related proteins using STITCH 5.0 revealed the following associations with pathways: response to stress; bacterium; and LPS (GO:0080134; 0009617; 0032496). Markedly, alpha-2-HS-glycoprotein (AHSG or fetuin-A) and lactotransferrin (LTF) potentially presented since the first hour of LPS stimulation, and were highly up-regulated at 24 h. Taken together, we reported proteomic profiling of vascular endothelial cell-specific DAMPs in response to early an in vitro LPS stimulation, suggesting that these early damage-response protein candidates could be novel early biomarkers associated with sepsis.

Neurovascular Hypoxia Trajectories Assessed by Photoacoustic Imaging in a Murine Model of Cardiac Arrest and Resuscitation.

Cardiac arrest is a common cause of death annually mainly due to postcardiac arrest syndrome that leads to multiple organ global hypoxia and dysfunction after resuscitation. The ability to quantify vasculature changes and tissue oxygenation is crucial to adapt patient treatment in order to minimize major outcomes after resuscitation. For the first time, we applied high-resolution ultrasound associated with photoacoustic imaging (PAI) to track neurovascular oxygenation and cardiac function trajectories in a murine model of cardiac arrest and resuscitation. We report the preservation of brain oxygenation is greater compared to that in peripheral tissues during the arrest. Furthermore, distinct patterns of cerebral oxygen decay may relate to the support of vital brain functions. In addition, we followed trajectories of cerebral perfusion and cardiac function longitudinally after induced cardiac arrest and resuscitation. Volumetric cerebral oxygen saturation (sO2) decreased 24 h postarrest, but these levels rebounded at one week. However, systolic and diastolic cardiac dysfunction persisted throughout and correlated with cerebral hypoxia. Pathophysiologic biomarker trends, identified via cerebral PAI in preclinical models, could provide new insights into understanding the pathophysiology of cardiac arrest and resuscitation.

Neurovascular hypoxia after mild traumatic brain injury in juvenile mice correlates with heart-brain dysfunctions in adulthood.

AIM: Retrospective studies suggest that mild traumatic brain injury (mTBI) in pediatric patients may lead to an increased risk of cardiac events. However, the exact functional and temporal dynamics and the associations between heart and brain pathophysiological trajectories are not understood. METHODS: A single impact to the left somatosensory cortical area of the intact skull was performed on juvenile mice (17 days postnatal). Cerebral 3D photoacoustic imaging was used to measure the oxygen saturation (sO2 ) in the impacted area 4 h after mTBI followed by 2D and 4D echocardiography at days 7, 30, 90, and 190 post-impact. At 8 months, we performed a dobutamine stress test to evaluate cardiac function. Lastly, behavioral analyses were conducted 1 year after initial injury. RESULTS: We report a rapid and transient decrease in cerebrovascular sO2 and increased hemoglobin in the impacted left brain cortex. Cardiac analyses showed long-term diastolic dysfunction and a diminished systolic strain response under stress in the mTBI group. At the molecular level, cardiac T-p38MAPK and troponin I expression was pathologic modified post-mTBI. We found linear correlations between brain sO2 measured immediately post-mTBI and long-term cardiac strain after 8 months. We report that initial cerebrovascular hypoxia and chronic cardiac dysfunction correlated with long-term behavioral changes hinting at anxiety-like and memory maladaptation. CONCLUSION: Experimental juvenile mTBI induces time-dependent cardiac dysfunction that corresponds to the initial neurovascular sO2 dip and is associated with long-term behavioral modifications. These imaging biomarkers of the heart-brain axis could be applied to improve clinical pediatric mTBI management.

pH-sensitive doxorubicin-tocopherol succinate prodrug encapsulated in docosahexaenoic acid-based nanostructured lipid carriers: An effective strategy to improve pharmacokinetics and reduce toxic effects.

Side effects often limit the use of doxorubicin (DOX) in cancer treatment. We have recently developed a nanostructured lipid carrier (NLC) formulation for synergistic chemotherapy, encapsulating DOX and the anticancer adjuvants docosahexaenoic acid (DHA) and α-tocopherol succinate (TS). Hydrophobic ion-pairing with TS allowed a high DOX entrapment in the nanocarrier. In this work, we investigated the pharmacokinetics of this formulation after intravenous administration in mice. The first data obtained led us to propose synthesizing covalent DOX-TS conjugates to increase DOX retention in the NLC. We successfully conjugated DOX to TS via an amide or hydrazone bond. In vitro studies in 4T1 tumor cells indicated low cytotoxicity of the amide derivative, while the hydrazone conjugate was effective in killing cancer cells. We encapsulated the hydrazone derivative in a DHA-based nanocarrier (DOX-hyd-TS/NLC), which had reduced particle size and high drug encapsulation efficiency. The pH-sensitive hydrazone bond allowed controlled DOX release from the NLC, with increased drug release at acidic conditions. In vivo studies revealed that DOX-hyd-TS/NLC had a better pharmacokinetic profile than free DOX and attenuated the short-term cardiotoxic effects caused by DOX, such as QT prolongation and impaired left ventricular systolic function. Moreover, this formulation showed excellent therapeutic performance by reducing tumor growth in 4T1 tumor-bearing mice and decreasing DOX-induced toxicity to the heart and liver, demonstrated by hematologic, biochemical, and histologic analyses. These results indicate that DOX-hyd-TS/NLC may be a promising nanocarrier for breast cancer treatment.

Global 11 Beta-Hydroxysteroid Dehydrogenase Activity Assessed by the Circulating Cortisol to Cortisone Ratio is Associated with Features of Metabolic Syndrome.

Background: 11 Beta-hydroxysteroid dehydrogenases (11HSDs) are enzymes involved in the interconversion of cortisol and cortisone. There are two isoenzymes of 11HSD, 11HSD1 and 11HSD2. A causative role of 11HSD, particularly 11HSD1, in metabolic syndrome is well established in experimental animals. However, its role in human metabolic syndrome is less clear. We examined the influence of global 11HSD activity on metabolic syndrome in the general population, using the circulating cortisol:cortisone ratio as an index of global 11HSD activity. Methods: A subsample of 269 sera randomly selected from the Thai National Health Examination Survey IV samples was analyzed for serum cortisol and cortisone levels by liquid chromatography-tandem mass spectrometry. Results: There was no association between serum cortisol and age. However, circulating cortisone was negatively correlated with age (r = -0.12, P < 0.001), and the serum cortisol:cortisone ratio was positively associated with age (r = 0.03, P < 0.001). No association was found between serum cortisol:cortisone ratio and body mass index (BMI) or serum lipids. Multivariate analyses showed that the serum cortisol:cortisone ratio was associated with high blood pressure (P < 0.05) independent of age, BMI, and sex. In subjects without hypertension, the serum cortisol to cortisone ratio was associated with mean systolic blood pressure after controlling for age, BMI, and sex. The cortisol:cortisone ratio was not significantly different between subjects with and without diabetes. After excluding the 16 subjects with diabetes, it was found that the serum cortisol:cortisone ratio was positively associated with fasting plasma glucose independent of age, BMI, and sex (P < 0.01). Conclusions: The global index of 11HSD activity, assessed by the circulating cortisol:cortisone ratio, was related to high blood pressure and fasting plasma glucose and may serve as a proxy to global 11HSD activity.

Independent and Opposite Associations Between Branched-Chain Amino Acids and Lysophosphatidylcholines With Incident Diabetes in Thais.

Branched-chain amino acids (BCAAs) and lysophosphatidylcholines (LPCs) have been reported to be associated with diabetes. The purpose of the present study was to investigate the relative contributions of BCAAs and LPCs to the progression of prediabetes to diabetes using a targeted metabolomic approach. This study was part of a health survey of employees of the Electricity Generating Authority of Thailand (n = 79; nine females and 70 males). A targeted metabolomics analysis was performed using an AbsoluteIDQ® p180 kit, flow injection analysis, and liquid chromatography-tandem mass spectrometry. The highest variable importance in projection (VIP) scores for the progression to diabetes of the amino acids and phospholipids were associated with isoleucine and LPC acyl C28:1, respectively. Using logistic regression analysis, we found that high baseline isoleucine concentration was associated with a higher incidence of diabetes, while high LPC acyl 28:1 was associated with a lower incidence. Isoleucine and LPC acyl 28:1 were independently associated with incident diabetes in a model that also included conventional risk factors for diabetes (baseline fasting plasma glucose (FPG), age, sex, and body mass index (BMI)). In addition, isoleucine and LPC acyl 28:1 were independently associated with serum HbA1c 5 years later in a robust regression model that also included baseline FPG, age, sex, and BMI. Isoleucine, LPC acyl 28:1, age, and FPG were significantly associated with HbA1c at this time. In conclusion, these results provide evidence that isoleucine and LPC acyl C28:1 have respective positive and negative independent associations with incident diabetes.

Experimental Myocardial Infarction Elicits Time-Dependent Patterns of Vascular Hypoxia in Peripheral Organs and in the Brain.

Aims: Microvascular alterations occurring after myocardial infarction (MI) may represent a risk factor for multi-organ failure. Here we used in vivo photoacoustic (PA) imaging to track and define the changes in vascular oxygen saturation (sO2) occurring over time after experimental MI in multiple peripheral organs and in the brain. Methods and Results: Experimental MI was obtained in BALB/c mice by permanent ligation of the left anterior descending artery. PA imaging (Vevo LAZR-X) allowed tracking mouse-specific sO2 kinetics in the cardiac left ventricular (LV) anterior wall, brain, kidney, and liver at 4 h, 1 day, and 7 days post-MI. Here we reported a correlation between LV sO2 and longitudinal anterior myocardial strain after MI (r = -0.44, p < 0.0001, n = 96). Acute LV dysfunction was associated with global hypoxia, specifically a decrease in sO2 level in the brain (-5.9%), kidney (-6.4%), and liver (-7.3%) at 4 and 24 h post-MI. Concomitantly, a preliminary examination of capillary NG2DsRed pericytes indicated cell rarefication in the heart and kidney. While the cardiac tissue was persistently impacted, sO2 levels returned to pre-MI levels in the brain and in peripheral organs 7 days after MI. Conclusions: Collectively, our data indicate that experimental MI elicits precise trajectories of vascular hypoxia in peripheral organs and in the brain. PA imaging enabled the synchronous tracking of oxygenation in multiple organs and occurring post-MI, potentially enabling a translational diagnostic modality for the identification of vascular modifications in this disease setting.