Extracorporeal life support without systemic anticoagulation: a nitric oxide-based non-thrombogenic circuit for the artificial placenta in an ovine model.

BACKGROUND: Clinical translation of the extracorporeal artificial placenta (AP) is impeded by the high risk for intracranial hemorrhage in extremely premature newborns. The Nitric Oxide Surface Anticoagulation (NOSA) system is a novel non-thrombogenic extracorporeal circuit. This study aims to test the NOSA system in the AP without systemic anticoagulation. METHODS: Ten extremely premature lambs were delivered and connected to the AP. For the NOSA group, the circuit was coated with DBHD-N2O2/argatroban, 100 ppm nitric oxide was blended into the sweep gas, and no systemic anticoagulation was given. For the Heparin control group, a non-coated circuit was used and systemic anticoagulation was administered. RESULTS: Animals survived 6.8 ± 0.6 days with normal hemodynamics and gas exchange. Neither group had any hemorrhagic or thrombotic complications. ACT (194 ± 53 vs. 261 ± 86 s; p < 0.001) and aPTT (39 ± 7 vs. 69 ± 23 s; p < 0.001) were significantly lower in the NOSA group than the Heparin group. Platelet and leukocyte activation did not differ significantly from baseline in the NOSA group. Methemoglobin was 3.2 ± 1.1% in the NOSA group compared to 1.6 ± 0.6% in the Heparin group (p < 0.001). CONCLUSIONS: The AP with the NOSA system successfully supported extremely premature lambs for 7 days without significant bleeding or thrombosis. IMPACT: The Nitric Oxide Surface Anticoagulation (NOSA) system provides effective circuit-based anticoagulation in a fetal sheep model of the extracorporeal artificial placenta (AP) for 7 days. The NOSA system is the first non-thrombogenic circuit to consistently obviate the need for systemic anticoagulation in an extracorporeal circuit for up to 7 days. The NOSA system may allow the AP to be implemented clinically without systemic anticoagulation, thus greatly reducing the intracranial hemorrhage risk for extremely low gestational age newborns. The NOSA system could potentially be applied to any form of extracorporeal life support to reduce or avoid systemic anticoagulation.

The effect of air exposure on leucocyte and cytokine activation in an in-vitro model of cardiotomy suction.

INTRODUCTION: Cardiopulmonary bypass (CPB) is known to cause a systemic inflammatory and immune response. OBJECTIVE: An in-vitro model of cardiotomy suction was designed to quantify the effects of incrementally increased air-blood exposure on leucocyte marker CD11b and cytokine activation in two common anticoagulants, heparin and citrate. METHODS: Fresh human blood was exposed to increasing amounts of air flow for ten minutes. Leucocyte and cytokine levels were measured prior to and after ten minutes of air flow. Cytokine levels were also measured after air exposure when incubated for 24 hours at 37oC. RESULTS: Leucocyte activation, measured by CD11b, was elevated between baseline and air flow rates up to 50 mL/min. After 10 minutes of air exposure, no measured cytokine levels were elevated. After 24 hours of incubation, cytokine levels of TNFα, IL-10, IL-6, and IL-8 were elevated. However, only IL-8 was significantly elevated in citrated blood, but not in heparinized blood, when compared to baseline samples that were also incubated for 24 hours. CONCLUSION: This study investigates CD11b levels in response to an air stimulus in blood that was anticoagulated with citrate or heparin. Exposure to an air stimulus activates leucocytes. Activation of CD11b was less when using heparin as an anticoagulant compared to citrate. Cytokine activation occurs with air stimulation, but levels do not immediately rise, indicating that time is required to generate free cytokines.

Portable Nitric Oxide (NO) Generator Based on Electrochemical Reduction of Nitrite for Potential Applications in Inhaled NO Therapy and Cardiopulmonary Bypass Surgery.

A new portable gas phase nitric oxide (NO) generator is described for potential applications in inhaled NO (INO) therapy and during cardiopulmonary bypass (CPB) surgery. In this system, NO is produced at the surface of a large-area mesh working electrode by electrochemical reduction of nitrite ions in the presence of a soluble copper(II)-ligand electron transfer mediator complex. The NO generated is then transported into gas phase by either direct purging with nitrogen/air or via circulating the electrolyte/nitrite solution through a gas extraction silicone fiber-based membrane-dialyzer assembly. Gas phase NO concentrations can be tuned in the range of 5-1000 ppm (parts per million by volume for gaseous species), in proportion to a constant cathodic current applied between the working and counter electrodes. This new NO generation process has the advantages of rapid production times (5 min to steady-state), high Faraday NO production efficiency (ca. 93%), excellent stability, and very low cost when using air as the carrier gas for NO (in the membrane dialyzer configuration), enabling the development of potentially portable INO devices. In this initial work, the new system is examined for the effectiveness of gaseous NO to reduce the systemic inflammatory response (SIR) during CPB, where 500 ppm of NO added to the sweep gas of the oxygenator or to the cardiotomy suction air in a CPB system is shown to prevent activation of white blood cells (granulocytes and monocytes) during extracorporeal circulation with cardiotomy suction conducted with five pigs.

Improved in vivo performance of amperometric oxygen (PO2) sensing catheters via electrochemical nitric oxide generation/release.

A novel electrochemically controlled release method for nitric oxide (NO) (based on electrochemical reduction of nitrite ions) is combined with an amperometric oxygen sensor within a dual lumen catheter configuration for the continuous in vivo sensing of the partial pressure of oxygen (PO2) in blood. The on-demand electrochemical NO generation/release method is shown to be fully compatible with amperometric PO2 sensing. The performance of the sensors is evaluated in rabbit veins and pig arteries for 7 and 21 h, respectively. Overall, the NO releasing sensors measure both venous and arterial PO2 values more accurately with an average deviation of -2 ± 11% and good correlation (R(2) = 0.97) with in vitro blood measurements, whereas the corresponding control sensors without NO release show an average deviation of -31 ± 28% and poor correlation (R(2) = 0.43) at time points >4 h after implantation in veins and >6 h in arteries. The NO releasing sensors induce less thrombus formation on the catheter surface in both veins and arteries (p < 0.05). This electrochemical NO generation/release method could offer a new and attractive means to improve the biocompatibility and performance of implantable chemical sensors.

Blood biocompatibility of surface-bound multi-walled carbon nanotubes.

Blood clots when it contacts foreign surfaces following platelet activation. This can be catastrophic in clinical settings involving extracorporeal circulation such as during heart-lung bypass where blood is circulated in polyvinyl chloride tubing. Studies have shown, however, that surface-bound carbon nanotubes may prevent platelet activation, the initiator of thrombosis. We studied the blood biocompatibility of polyvinyl chloride, surface-modified with multi-walled carbon nanotubes in vitro and in vivo. Our results show that surface-bound multi-walled carbon nanotubes cause platelet activation in vitro and devastating thrombosis in an in vivo animal model of extracorporeal circulation. The mechanism of the pro-thrombotic effect likely involves direct multi-walled carbon nanotube-platelet interaction with Ca(2+)-dependant platelet activation. These experiments provide evidence, for the first time, that modification of surfaces with nanomaterials modulates blood biocompatibility in extracorporeal circulation.

Antithrombotic and Antimicrobial Potential of S-Nitroso-1-Adamantanethiol-Impregnated Extracorporeal Circuit.

This study presents the utilization of a novel, highly lipophilic nitric oxide (NO) donor molecule, S-nitroso-1-adamantanethiol (SNAT), for developing an NO-emitting polymer surface aimed at preventing thrombus formation and bacterial infection in extracorporeal circuits (ECCs). S-nitroso-1-adamantanethiol, a tertiary nitrosothiol-bearing adamantane species, was synthesized, characterized, and used to impregnate polyvinyl chloride (PVC) tubing for subsequent in vivo evaluation. The impregnation process with SNAT preserved the original mechanical strength of the PVC. In vitro assessments revealed sustained NO release from the SNAT-impregnated PVC tubing (iSNAT), surpassing or matching endothelial NO release levels for up to 42 days. The initial NO release remained stable even after 1 year of storage at -20°C. The compatibility of iSNAT with various sterilization techniques (OPA Plus, hydrogen peroxide, EtO) was tested. Acute in vivo experiments in a rabbit model demonstrated significantly reduced thrombus formation in iSNAT ECCs compared with controls, indicating the feasibility of iSNAT to mitigate coagulation system activation and potentially eliminate the need for systemic anticoagulation. Moreover, iSNAT showed substantial inhibition of microbial biofilm formation, highlighting its dual functionality. These findings underscore the promising utility of iSNAT for long-term ECC applications, offering a multifaceted approach to enhancing biocompatibility and minimizing complications.

Nicorandil attenuates ventricular dysfunction and organ injury after cardiopulmonary bypass.

BACKGROUND: Nicorandil, an adenosine triphosphate-sensitive potassium channel agonist and nitric oxide donor, is a coronary vasodilator used to treat ischemia-induced chest pain, but it's potential cardioprotective benefits during open heart surgery have not been thoroughly investigated. The study objective was to assess the impact of nicorandil on postoperative ventricular dysfunction and end-organ injury in an established experimental model of open-heart surgery with cardiopulmonary bypass (CPB) and cardioplegic arrest. We hypothesized that nicorandil would attenuate myocardial ischemia-reperfusion (IR) injury, preserve ventricular function, and reduce end-organ injury. METHODS: Rabbits were cannulated for CPB, followed by 60 min of aortic cross-clamp (ACC) with cold cardioplegic arrest, and 120 min of recovery after ACC removal. Nicorandil (or normal saline vehicle) was given intravenously 5 min before ACC and continued throughout the recovery period. Left ventricular developed pressure (LVDP), systolic contractility (LV + dP/dt), and diastolic relaxation (LV -dP/dt) were continuously recorded, and blood and tissue samples were collected for measurement of oxidant stress (OS), inflammation, apoptosis, and organ injury. RESULTS: Nicorandil significantly attenuated IR-induced LV dysfunction compared to saline control (R-120: LV + dP/dt: 1596 ± 397 vs. 514 ± 269 mmHg/s, p = 0.010; LV -dP/dt: -1524 ± 432 vs. -432 ± 243 mmHg/s, p < 0.001; LVDP: 55 ± 11 vs. 22 ± 5 mmHg, p = 0.046). Furthermore, nicorandil inhibited IR-induced increases in OS, inflammation, apoptosis, and organ injury. CONCLUSIONS: Nicorandil exhibits myocardial protection by attenuation of IR-induced LV dysfunction associated with OS, inflammation, apoptosis, and organ injury. Nicorandil should be explored further as a potential therapeutic strategy for limiting global IR injury during open-heart surgery in humans.

Measurement of tear glucose levels with amperometric glucose biosensor/capillary tube configuration.

An amperometric needle-type electrochemical glucose sensor intended for tear glucose measurements is described and employed in conjunction with a 0.84 mm i.d. capillary tube to collect microliter volumes of tear fluid. The sensor is based on immobilizing glucose oxidase on a 0.25 mm o.d. platinum/iridium (Pt/Ir) wire and anodically detecting the liberated hydrogen peroxide from the enzymatic reaction. Inner layers of Nafion and an electropolymerized film of 1,3-diaminobenzene/resorcinol greatly enhance the selectivity for glucose over potential interferences in tear fluid, including ascorbic acid and uric acid. Further, the new sensor is optimized to achieve very low detection limits of 1.5 ± 0.4 µM of glucose (S/N = 3) that is required to monitor glucose levels in tear fluid with a glucose sensitivity of 0.032 ± 0.02 nA/µM (n = 6). Only 4-5 µL of tear fluid in the capillary tube is required when the needle sensor is inserted into the capillary. The glucose sensor was employed to measure tear glucose levels in anesthetized rabbits over an 8 h period while also measuring the blood glucose values. A strong correlation between tear and blood glucose levels was found, suggesting that measurement of tear glucose is a potential noninvasive substitute for blood glucose measurements, and the new sensor configuration could aid in conducting further research in this direction.

The Effects of the Combined Argatroban/Nitric Oxide-Releasing Polymer on Platelet Microparticle-Induced Thrombogenicity in Coated Extracorporeal Circuits.

Clotting, anticoagulation, platelet consumption, and poor platelet function are major factors in clinical extracorporeal circulation (ECC). We have shown that nitric oxide-releasing (NOReL) coatings prevent thrombosis in a rabbit model of ECC without systemic anticoagulation. Nitric oxide-releasing prevents platelet adhesion and activation, resulting in preserved platelet count and function. Previous work has shown that activated platelets form platelet-derived microparticles (PMPs). These experiments were designed to determine if PMPs can identify platelet function during ECC. The objective of this study is to investigate the effects of NOReL on platelet activation and PMP formation during ECC. Uncoated ECCs, including with and without systemic heparin, and NOReL-coated ECCs, including DBHD/N2O2 and argatroban (AG)/DBHD/N2O2-coated ECCs without systemic heparin, were tested in a 4-hour rabbit thrombogenicity model. Before and after ECC exposure, platelets were stimulated with collagen, and PMPs were measured using flow cytometry. The uncoated ECCs clotted within the first hour, while the NOReL-coated ECCs circulated for 4 hours. During pre-ECC blood exposure, platelets stimulated with collagen produced PMPs. With post-ECC exposure, platelets from uncoated circuits generated less PMPs than baseline (mean ± SDs: 23246 ± 3611 baseline vs. 1300 ± 523 uncoated post circuit, p = 0.018) when stimulated with collagen. However, platelets from the AG/DBHD/N2O2-coated ECCs generated a greater number of PMPs as baseline values (23246 ± 3611 baseline vs. 37040 ± 3263 AG/DBHD/N2O2 post 4 hours circuit, p = 0.023). Blood exposure during ECC results in platelet activation and clotting in uncoated ECCs. The remaining circulating platelets have lost function, as demonstrated by the low PMP formation in response to collagen. AG/DBHD/N2O2-coated ECCs prevented significant platelet activation and clotting, while DBHD/N2O2 trended towards prevention of platelet activation. In addition, function of the circulating platelets was preserved, as demonstrated by PMP formation in response to collagen. These results indicate that PMPs may be an important measure of platelet activation during ECC. Platelet-derived microparticles may provide a simplified way to measure platelet function during clinical ECC.

Polydiacetylene Liposome Microarray toward Facile Measurement of Platelet Activation in Whole Blood.

The necessity of a simple measurement of platelet activation has been increasing in clinical medicine to regulate the proper dose of the antiplatelet drugs for patients having clinical outcomes in acute situations such as angina pectoris, stroke, or peripheral vascular disease or procedures involving angioplasty or coronary thrombolysis. We developed a self-signaling polydiacetylene (PDA) liposome microarray to detect activated platelets from whole blood samples in a single step. A specific antibody, 9F9 antibody, to platelet-bound fibrinogen was selected and conjugated to the PDA liposome microarray to quantify the fibrinogen-bound platelets. The developed PDA liposome-9F9 microarray generated an intense fluorescence signal when activated platelets in whole blood were introduced and also successfully distinguished the reduced platelet activation in the presence of Tirofiban, a model antiplatelet drug. The results of this single-step benchtop assay incorporates simple, sensitive, and rapid attributes that can detect the extent of platelet activation prior to needed clinical procedures.

Comparison of Diazeniumdiolated Dialkylhexanediamines as Nitric Oxide Release Agents on Nonthrombogenicity in an Extracorporeal Circulation Model.

When blood from a patient is circulated through extracorporeal circuits (ECCs), such as in cardiopulmonary bypass or extracorporeal life support, platelets in the blood are activated and form a thrombus. This is prevented clinically with a range of different systemic anticoagulation agents (e.g., heparin); however, this increases a patient's risk of hemorrhage. Previous work with nitric oxide (NO) releasing materials using the combined diazeniumdiolated diamine, N-N-di-N'-butyl-1,6-hexanediamine (DBHD), and a polymer-linked thrombin inhibitor, argatroban (AG), showed significant nonthrombogenicity in ECCs using a 4 h rabbit model. Herein, we evaluated if diazeniumdiolated N-N-di-N'-propyl-1,6-hexanediamine (DPHDN2O2), which has a slightly lower degree of lipophilicity compared to DBHDN2O2, would provide similar nonthrombogenicity as the AG/DBHDN2O2-polymer-coated circuits. While DPHDN2O2 releases NO at a higher flux rate than DBHDN2O2 when coated (within CarboSil polymer) on the inner wall of polyvinyl chloride tubing, neither coated circuit significantly affected animal hemodynamics. Both diazeniumdiolated diamines, in combination with immobilized AG or alone, significantly reduced thrombus formation similarly in the 4 h rabbit model (vs uncoated control): AG/DBHDN2O2: 0.12 ± 0.03 cm2; DBHDN2O2: 2.57 ± 0.82 cm2; AG/DPHDN2O2: 0.68 ± 0.22 cm2; DPHDN2O2: 1.87 + 1.26 cm2; uncoated control: 6.95 ± 0.82 cm2. AG/DPHDN2O2 was no different than AG/DBHDN2O in preserving platelet count and function. In addition, AG did not leach into the systemic circulation as the total clotting times were insignificantly different from the baseline values (AG/DPHDN2O2: 12.7 + 0.5 s (n = 3); AG/DBHDN2O2: 12.3 + 0.7 s (n = 3); baseline: 13.9 + 0.3 s (n = 13)). Based on these results, both DPHDN2O2 and DPHDN2O2 are good candidates as NO donor molecules for creating nonthrombogenic polymer coatings for ECCs.

Normoxic re-oxygenation ameliorates end-organ injury after cardiopulmonary bypass.

BACKGROUND: In a rabbit model of cardiopulmonary bypass (CPB) and cardioplegic arrest, we previously showed that hyperoxic myocardial reperfusion was associated with increased left ventricular (LV) systolic dysfunction and myocardial injury compared with normoxic reperfusion. The aim of this study was to evaluate in our experimental model the impact of post-CPB reperfusion conditions on other organs potentially vulnerable to ischemic injury such as the brain and kidney. METHODS: After 60 min of CPB, aortic cross-clamp, and cold cardioplegic arrest, rabbits were reperfused under hyperoxic or normoxic conditions for 120 min. Left ventricular systolic contractility (LV + dP/dt) and diastolic relaxation (LV -dP/dt) were continuously recorded, and end-organ injury was assessed by measuring circulating biomarkers specific for kidney (cystatin C and creatinine) and brain injury [S100B and neuron specific enolase (NSE)]. At completion of the protocol, kidney and brain tissues were harvested for measuring oxidant stress (OS), inflammation and apoptosis. RESULTS: Following aortic cross-clamp removal, rabbits exposed to normoxic reperfusion demonstrated preserved LV systolic and diastolic function compared with hyperoxic reperfusion (LV + dP/dt: 70 ± 14% of pre-CPB vs. 36 ± 21%, p = 0.018; LV -dP/dt: 72 ± 36% of pre-CPB vs. 33 ± 20%, p = 0.023). Similarly, CPB increased plasma creatinine, S100B and NSE that were significantly attenuated by normoxic reperfusion compared with hyperoxic reperfusion (creatinine: 4.0 ± 0.5 vs. 7.1 ± 0.8 mg/dL, p = 0.004; S100B: 4.0 ± 0.8 vs. 6.7 ± 1.0 ng/mL, p = 0.047; NSE: 57.7 ± 6.8 vs. 101.3 ± 16.1 pg/mL, p = 0.040). Furthermore, both kidney and brain tissues showed increased mRNA expression and activation of pathways for OS, inflammation, and apoptosis, that were reduced under normoxic compared with hyperoxic conditions. CONCLUSIONS: Normoxic reperfusion ameliorates cardiac, renal and neural injury compared with hyperoxic reperfusion in an in vivo animal model of CPB and cardioplegic arrest. This protective effect of normoxic reperfusion may be due to a reduction in signaling pathways for OS, inflammation, and apoptosis.

Assessing and improving the biocompatibility of microfluidic artificial lungs.

Microfluidic artificial lungs (µALs) have the potential to improve the treatment and quality of life for patients with acute or chronic lung injury. In order to realize the full potential of this technology (including as a destination therapy), the biocompatibility of these devices needs to be improved to produce long-lasting devices that are safe for patient use with minimal or no systemic anticoagulation. Many studies exist which probe coagulation and thrombosis on polydimethyl siloxane (PDMS) surfaces, and many strategies have been explored to improve surface biocompatibility. As the field of µALs is young, there are few studies which investigate biocompatibility of functioning µALs; and even fewer which were performed in vivo. Here, we use both in vitro and in vivo models to investigate two strategies to improve µAL biocompatibility: 1) a hydrophilic surface coating (polyethylene glycol, PEG) to prevent surface fouling, and 2) the addition of nitric oxide (NO) to the sweep gas to inhibit platelet activation locally within the µAL. In this study, we challenge µALs with clottable blood or platelet-rich plasma (PRP) and monitor the resistance to blood flow over time. Device lifetime (the amount of time the µAL remains patent and unobstructed by clot) is used as the primary indicator of biocompatibility. This study is the first study to: 1) investigate the effect of NO release on biocompatibility in a microfluidic network; 2) combine a hydrophilic PEG coating with NO release to improve blood compatibility; and 3) perform extended in vivo biocompatibility testing of a µAL. We found that µALs challenged in vitro with PRP remained patent significantly longer when the sweep gas contained NO than without NO. In the in vivo rabbit model, neither approach alone (PEG coating nor NO sweep gas) significantly improved biocompatibility compared to controls (though with larger sample size significance may become apparent); while the combination of a PEG coating with NO sweep gas resulted in significant improvement of device lifetime. STATEMENT OF SIGNIFICANCE: The development of microfluidic artificial lungs (µALs) can potentially have a massive impact on the treatment of patients with acute and chronic lung impairments. Before these devices can be deployed clinically, the biocompatibility of µALs must be improved and more comprehensively understood. This work explores two strategies for improving biocompatibility, a hydrophilic surface coating (polyethylene glycol) for general surface passivation and the addition of nitric oxide (NO) to the sweep gas to quell platelet and leukocyte activation. These two strategies are investigated separately and as a combined device treatment. Devices are challenged with clottable blood using in vitro testing and in vivo testing in rabbits. This is the first study to our knowledge that allows statistical comparisons of biocompatible µALs in animals, a key step towards eventual clinical use.

A CCR2/CCR5 antagonist attenuates an increase in angiotensin II-induced CD11b+ monocytes from atherogenic ApoE-/- mice.

OBJECTIVE: Monocyte infiltration into the vessel wall, a process primarily mediated by the interaction between monocyte chemoattractant protein-1 (MCP-1) and its receptor, CCR2, is a key step in atherogenesis. Angiotensin II (Ang II) enhances this monocyte infiltration by increasing the endothelial binding integrin, CD11b. However, the modulation of the Ang II-induced CD11b expression in monocytes in not clear. The aim of this study was to determine if MCP-1/MCP-2 receptor (CCR2) interaction regulates monocyte CD11b expression after 7 days of Ang II infusion. METHODS AND RESULTS: In ApoE(-/-) mice continuous subcutaneous infusion of Ang II (0.75 mg/kg/day) for 7 days significantly increased CD11b expression in circulating monocytes as measured by flow cytometry. CD11b expression in ApoE(-/-) was increased from 135 +/- 9 to 176 +/- 12 mean fluorescent intensity (MFI), control and Ang II-treated, respectively while in C57B/J wildtype mice CD11b increased from 128 +/- 13 to 174 +/- 8 MFI, control and Ang II-treated, respectively. Interestingly, co-infusion of either MCP-1 neutralizing antibody (25 microg/kg/day) or a CCR2 antagonist (500 microg/kg/day) with Ang II for 7 days effectively inhibited monocyte CD11b expression and this inhibition was accompanied by a down-regulated vascular infiltration of Mac-2 positive monocyte-derived macrophages. CONCLUSION: Our data in the atherogenic ApoE(-/-) mouse demonstrates that the Ang II induced increase in both monocytic CD11b integrin expression and monocyte vascular infiltration occurs early in atherogenesis. These Ang II-induced monocytic changes are in part regulated through the MCP-1/CCR2 interaction.

Differential Effects of Normoxic and Hyperoxic Reperfusion on Global Myocardial Ischemia-Reperfusion Injury.

The objectives were to investigate if after hypoxia or ischemia, normoxic reperfusion is associated with less oxidant stress (OS), inflammation, and myocardial injury than hyperoxic reperfusion. In this study, cardiomyocytes (H9c2 cells) were cultured in hypoxia, followed by reoxygenation in normoxia or hyperoxia. Cardiomyocyte OS, inflammation, and apoptosis were measured. In parallel experiments, rabbits were cannulated for cardiopulmonary bypass (CPB). Following cardioplegic arrest and aortic cross-clamp removal, hearts were reperfused under normoxic or hyperoxic conditions. Left ventricular developed pressure and contractility (LV +dP/dt) were recorded, and blood samples and heart tissues were collected for measurement of OS, inflammation, and cardiac injury. Results showed that H9c2 cells exposed to hyperoxic reoxygenation showed significant increases in OS, inflammation, and apoptosis compared to normoxic reoxygenation. Following CPB and 2-hour hyperoxic reperfusion, LV +dP/dt and left ventricular developed pressure were significantly decreased compared with pre-CPB values (to 36 ± 21%, P = 0.002; and 53 ± 20%, P = 0.02, respectively), associated with significant increases in all plasma and tissue biomarkers for OS, inflammation, and myocardial injury. In contrast, LV +dP/dt was relatively well preserved under normoxic reperfusion conditions (to 70 ± 14% after 2-hour reperfusion), and was associated with an attenuated myocardial OS, inflammatory, apoptotic, and injury response compared to the hyperoxia group (eg, cTn-I: 5.9 ± 1.5 vs 20.2 ± 7.6 ng/mL, respectively, P < 0.0001). Overall, in both in vitro and in vivo experiments, normoxic reperfusion/reoxygenation was associated with less robust OS, inflammation, apoptosis, and myocardial injury compared with hyperoxic reperfusion/reoxygenation. These results suggest that hyperoxia should be avoided to minimize myocardial OS, inflammation, and ventricular dysfunction after CPB.

The T- and L-type calcium channel blocker (CCB) mibefradil attenuates leg edema induced by the L-type CCB nifedipine in the spontaneously hypertensive rat: a novel differentiating assay.

Among the L-type calcium channel blockers (CCBs), particularly dihydropyridines like nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], a common adverse effect is vasodilatory edema. Newer CCBs, such as the T- and L-type CCB, mibefradil [(1S,2S)-2-[2[[3-(2-benzimidazolylpropyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate], demonstrate antihypertensive efficacy similar to that of their predecessors but seem to have a reduced propensity to cause edema. Using a magnetic resonance imaging (MRI) T(2) mapping technique, we investigated the ability of mibefradil to reduce extracellular water accumulation caused by the L-type CCB, nifedipine, in the hindleg skeletal muscle of the spontaneously hypertensive rat. Mibefradil (10 mg/kg i.v.) and nifedipine (1 mg/kg i.v.) lowered mean arterial blood pressure by 97 +/- 5 and 77 +/- 4 mm Hg, respectively. MRI edema index (expressed as percentage increase of integral T(2) over predrug control) was significantly higher with nifedipine (2606 +/- 86%; p < 0.05) than with mibefradil (981 +/- 171%) measured 30 to 60 min after the start of drug infusion. The hindleg edema caused by nifedipine was dose dependently decreased by coadministration of mibefradil (0, 0.3, or 3 mg/kg). The hindleg edema formation was not due to albumin leakage into the interstitial space based on immunostaining. However, a 4.2-fold increase in the arterial L-/T-type CC mRNA expression ratio was observed compared with the venous L/T ratio as shown by quantitative reverse transcription polymerase chain reaction. These results demonstrate the novel utility of MRI to measure extravascular water after acute exposure to CCBs and indicate that T-type CCB activity may reduce L-type CCB-induced vasodilatory edema in the skeletal muscle vasculature, possibly by a differential effect on arteriole and venule dilatation.

A nonpeptide, piperidine renin inhibitor provides renal and cardiac protection in double-transgenic mice expressing human renin and angiotensinogen genes.

INTRODUCTION: Controlling hypertension by angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB), mechanisms that inhibit later pathway steps in the renin-angiotensin system (RAS), have clinically afforded protection against cardiac and renal disease. MATERIALS AND METHODS: In order to determine if blocking the RAS rate-limiting step of angiotensin II generation via renin inhibition could afford similar end organ protection in a human-relevant preclinical model, this study investigated the cardiac and renal effects of a nonpeptide, piperidine renin inhibitor (RI; 100 mg/kg/day PO) in double transgenic mice (dTGM) which express both human renin and angiotensinogen genes. RI was compared to the ARB, candesartan (3 mg/kg/day PO), and to the ACEI, enalapril (60 mg/kg/day PO) in a 4-week dosing paradigm. These doses of RI, ACEI and ARB were previously found to normalize mean blood pressure (MBP) to 110 + 3, 109 + 7 and 107 + 6 mmHg, respectively, after 1 day of treatment. RESULTS AND DISCUSSION: In the dTGM, PRA, plasma aldosterone, GFR, microalbuminuria and left ventricular free wall thickness (LVH) were higher than in the wild type C57BL/6 mice. Microalbuminuria and LVH were significantly reduced by 93% and 9% for the RI, 83% and 13% for enalapril and 73% and 6% for candesartan, respectively. PRA and aldosterone were reduced by the RI 56% and 23%, respectively. These results suggest that the RI provides protection against cardiac and renal disease, similar to ARB and ACEI.

Attenuation of Thrombin-Mediated Fibrin Formation via Changes in Fibrinogen Conformation Induced by Reaction with S-nitroso-N-acetylpenicillamine, but not S-nitrosoglutathione.

Previous work in a 4 h rabbit thrombogenicity model has shown that a nitric oxide- (NO) generating polymer extracorporeal circuits (ECC) with infusion of S-nitroso-N-acetyl-penicillamine (SNAP) preserved platelets eventhough platelets were activated as shown by an increase in the glycoprotein, p-selectin. The platelet preservation mechanism was shown to be due to a changing fibrinogen structure leading to attenuation of platelet aggregation. Understanding the effects that SNAP, another RSNO, S-nitroso-glutathione (GSNO) as well as the non-RSNO, sodium nitroprusside (SNP), may have on human fibrinogen polymerization, this in vitro study evaluated the released NO effects on the thrombin-mediated fibrin formation and fibrinogen structure. Thrombin-induced fibrin formation at 300 µM SNAP (50 + 11% of baseline) was significantly reduced compared to SNAP's parent, N-acetyl-penicillamine (NAP) (95 + 13%) after 1 h of RSNO exposure. GSNO, its parent, glutathione (GSH) and 1000 ppm NO gas did not attenuate the thrombin-mediated fibrin formation. SNAP, NAP and SNP exposure for 1 h, however, did not decrease thrombin activity by directly inhibiting thrombin itself. Changes in fibrinogen conformation as measured by intrinsic tryptophan fluorescence significantly decreased in the 300 µM SNAP (38057 + 1196 mean fluorescence intensity (MFI) and SNP (368617 + 541 MFI) groups versus the NAP control (47937 + 1196 MFI). However, infused 1000 ppm NO gas had no direct effect on the ITF after 1 h incubation at 37°C. High performance liquid chromatography (HPLC) showed that fibrinogen degradation by 0.03 U/ml thrombin was concentration-dependently reduced after 1 h with SNAP but not with NAP or SNP. Western blotting showed RSNOs, SNAP, NAP and the non-RSNO, SNP-incubated fibrinogen solutions showed that the percent level of the Aγ dimer to total Aγ dimer + γ monomer was significantly reduced in the case of the SNAP group when compared to SNP group. These results suggest that NO donors such as SNAP and SNP induce fibrinogen conformational changes by potentially nitrosating fibrinogen tyrosine residues. These NO-mediated fibrinogen changes induced via NO donors may provide another mechanism of NO for improving thromboresistance in ECC.

Circulating succinate is elevated in rodent models of hypertension and metabolic disease.

BACKGROUND: Recent evidence suggests that succinate, long known as an intermediate in the citric acid cycle, may also have a role as a signaling molecule through GPR91 and that activation of this receptor results in blood pressure (BP) elevation via the renin-angiotensin system. We sought to test the hypothesis that GPR91 contributes to BP elevation in hypertension. In addition we investigated whether elevated succinate in diabetes could contribute to the increased rate of gluconeogenesis in that condition. METHODS: Circulating succinate concentration was measured using liquid chromatography tandem mass spectrometry in rodent models of hypertension and metabolic disease as well as in human hypertensives and type 2 diabetics in comparison to control subjects. RESULTS: Elevated succinate was detected in spontaneously hypertensive rats (SHR), ob/ob mice, db/db mice, and fa/fa rats in comparison to their non-diseased controls. The changes in concentration are consistent with activation of GPR91. In contrast, neither human hypertensives nor diabetic patients had elevated succinate in comparison to controls. CONCLUSIONS: These findings are consistent with a role of GPR91 signaling in rodent hypertension and diabetes models but not in the analogous human diseases.

Reduction of Thrombosis and Bacterial Infection via Controlled Nitric Oxide (NO) Release from S-Nitroso-N-acetylpenicillamine (SNAP) Impregnated CarboSil Intravascular Catheters.

Nitric oxide (NO) has many important physiological functions, including its ability to inhibit platelet activation and serve as potent antimicrobial agent. The multiple roles of NO in vivo have led to great interest in the development of biomaterials that can deliver NO for specific biomedical applications. Herein, we report a simple solvent impregnation technique to incorporate a nontoxic NO donor, S-nitroso-N-acetylpenicillamine (SNAP), into a more biocompatible biomedical grade polymer, CarboSil 20 80A. The resulting polymer-crystal composite material yields a very stable, long-term NO release biomaterial. The SNAP impregnation process is carefully characterized and optimized, and it is shown that SNAP crystal formation occurs in the bulk of the polymer after solvent evaporation. LC-MS results demonstrate that more than 70% of NO release from this new composite material originates from the SNAP embedded CarboSil phase, and not from the SNAP species leaching out into the soaking solution. Catheters prepared with CarboSil and then impregnated with 15 wt % SNAP provide a controlled NO release over a 14 d period at physiologically relevant fluxes and are shown to significantly reduce long-term (14 day) bacterial biofilm formation against Staphylococcus epidermidis and Pseudonomas aeruginosa in a CDC bioreactor model. After 7 h of catheter implantation in the jugular veins of rabbit, the SNAP CarboSil catheters exhibit a 96% reduction in thrombus area (0.03 ± 0.01 cm2/catheter) compared to the controls (0.84 ± 0.19 cm2/catheter) (n = 3). These results suggest that SNAP impregnated CarboSil can become an attractive new biomaterial for use in preparing intravascular catheters and other implanted medical devices.