The Effect of Fluid Pre-loading on Vital Signs and Hemodynamic Parameters in a Porcine Model of Lipopolysaccharide-Induced Endotoxemia.

Background Animal models of distributive hypotension and resuscitation allow the assessment of hemodynamic monitoring modalities and resuscitation strategies. The fluid-first paradigm for resuscitation is currently being challenged with clinical trials. In this investigation, venous return and perfusion are assessed, and full hemodynamics are characterized, in a porcine model of endotoxemic hypotension with and without fluid pre-loading. Methods Two groups of six pigs had the induction of standardized endotoxemic hypotension ("critical hypotension"). Group 1 underwent four 10 cc/kg crystalloid boluses, and Group 2 was not fluid pre-resuscitated. Both groups underwent progressive norepinephrine (NE) up-titration to 0.25 mcg/kg/minute over 30 minutes. Vital signs, central parameters, and laboratory values were obtained at baseline, "critical hypotension," after each bolus and during NE administration. Results Endotoxemia decreased the systemic vascular resistance (SVR) in Group 1 (1031±106 dyn/s/cm-5 versus 738±258 dyn/s/cm-5; P=0.03) and Group 2 (1121±196 dyn/s/cm-5 versus 759±342 dyn/s/cm-5; P=0.003). In Group 1, the four fluid boluses decreased heart rate (HR), pulmonary capillary wedge pressure (PCWP), and central venous pressure (CVP) (P<0.05). No changes were observed in blood pressure, cardiac output (CO), or lactate. NE up-titration increased HR in Group 1 and decreased CVP in both groups. Higher final CVP (11 {3} versus 4 {4} mmHg; P=0.01) and PCWP (5 {1} versus 2 {2} mmHg; P=0.005) values were observed in Group 1 relative to Group 2, reflecting increased venous return. Conclusions Porcine endotoxemic hypotension and resuscitation were robustly characterized. In this model, fluid loading improved venous return with NE, though perfusion (CO) was preserved by increased NE-induced chronotropy.

Central Venous Waveform Analysis and Cardiac Output in a Porcine Model of Endotoxemic Hypotension and Resuscitation.

BACKGROUND: Cardiac output (CO) is a valuable proxy for perfusion, and governs volume responsiveness during resuscitation from distributive shock. The underappreciated venous system has nuanced physiology that confers valuable hemodynamic information. In this investigation, deconvolution of the central venous waveform by the fast Fourier transformation (FFT) algorithm is performed to assess its ability to constitute a CO surrogate in a porcine model of endotoxemia-induced distributive hypotension and resuscitation. STUDY DESIGN: Ten pigs were anesthetized, catheterized, and intubated. A lipopolysaccharides infusion protocol was used to precipitate low systemic vascular resistance hypotension. Four crystalloid boluses (10 cc/kg) were then given in succession, after which heart rate, mean arterial pressure, thermodilution-derived CO, central venous pressure (CVP), and the central venous waveform were collected, the last undergoing fast Fourier transformation analysis. The amplitude of the fundamental frequency of the central venous waveform's cardiac wave (f0-CVP) was obtained. Heart rate, mean arterial pressure, CVP, f0-CVP, and CO were plotted over the course of the boluses to determine whether f0-CVP tracked with CO better than the vital signs, or than CVP itself. RESULTS: Distributive hypotension to a 25% mean arterial pressure decrement was achieved, with decreased systemic vascular resistance (mean 918 ± 227 [SD] dyne/s/cm-5 vs 685 ± 180 dyne/s/cm-5; p = 0.038). Full hemodynamic parameters characterizing this model were reported. Slopes of linear regression lines of heart rate, mean arterial pressure, CVP, f0-CVP, and CO were -2.8, 1.7, 1.8, 0.40, and 0.35, respectively, demonstrating that f0-CVP values closely track with CO over the 4-bolus range. CONCLUSIONS: Fast Fourier transformation analysis of the central venous waveform may allow real-time assessment of CO during resuscitation from distributive hypotension, possibly offering a venous-based approach to clinical estimation of volume responsiveness.

Blocking P2X7 receptor with AZ 10606120 exacerbates vascular hyperpermeability and inflammation in murine polymicrobial sepsis.

Sepsis is a devastating disease with high morbidity and mortality and no specific treatments. The pathophysiology of sepsis involves a hyperinflammatory response and release of damage-associated molecular patterns (DAMPs), including adenosine triphosphate (ATP), from activated and dying cells. Purinergic receptors activated by ATP have gained attention for their roles in sepsis, which can be pro- or anti-inflammatory depending on the context. Current data regarding the role of ATP-specific purinergic receptor P2X7 (P2X7R) in vascular function and inflammation during sepsis are conflicting, and its role on the endothelium has not been well characterized. In this study, we hypothesized that the P2X7R antagonist AZ 10606120 (AZ106) would prevent endothelial dysfunction during sepsis. As proof of concept, we first demonstrated the ability of AZ106 (10 µM) to prevent endothelial dysfunction in intact rat aorta in response to IL-1ß, an inflammatory mediator upregulated during sepsis. Likewise, blocking P2X7R with AZ106 (10 µg/g) reduced the impairment of endothelial-dependent relaxation in mice subjected to intraperitoneal injection of cecal slurry (CS), a model of polymicrobial sepsis. However, contrary to our hypothesis, AZ106 did not improve microvascular permeability or injury, lung apoptosis, or illness severity in mice subjected to CS. Instead, AZ106 elevated spleen bacterial burden and circulating inflammatory markers. In conclusion, antagonism of P2X7R signaling during sepsis appears to disrupt the balance between its roles in inflammatory, antimicrobial, and vascular function.

Therapeutic MK2 inhibition blocks pathological vascular smooth muscle cell phenotype switch.

Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP-treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype-associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.

Hemodynamic Parameters in the Assessment of Fluid Status in a Porcine Hemorrhage and Resuscitation Model.

BACKGROUND: Measuring fluid status during intraoperative hemorrhage is challenging, but detection and quantification of fluid overload is far more difficult. Using a porcine model of hemorrhage and over-resuscitation, it is hypothesized that centrally obtained hemodynamic parameters will predict volume status more accurately than peripherally obtained vital signs. METHODS: Eight anesthetized female pigs were hemorrhaged at 30 ml/min to a blood loss of 400 ml. After each 100 ml of hemorrhage, vital signs (heart rate, systolic blood pressure, mean arterial pressure, diastolic blood pressure, pulse pressure, pulse pressure variation) and centrally obtained hemodynamic parameters (mean pulmonary artery pressure, pulmonary capillary wedge pressure, central venous pressure, cardiac output) were obtained. Blood volume was restored, and the pigs were over-resuscitated with 2,500 ml of crystalloid, collecting parameters after each 500-ml bolus. Hemorrhage and resuscitation phases were analyzed separately to determine differences among parameters over the range of volume. Conformity of parameters during hemorrhage or over-resuscitation was assessed. RESULTS: During the course of hemorrhage, changes from baseline euvolemia were observed in vital signs (systolic blood pressure, diastolic blood pressure, and mean arterial pressure) after 100 ml of blood loss. Central hemodynamic parameters (mean pulmonary artery pressure and pulmonary capillary wedge pressure) were changed after 200 ml of blood loss, and central venous pressure after 300 ml of blood loss. During the course of resuscitative volume overload, changes were observed from baseline euvolemia in mean pulmonary artery pressure and central venous pressure after 500-ml resuscitation, in pulmonary capillary wedge pressure after 1,000-ml resuscitation, and cardiac output after 2,500-ml resuscitation. In contrast to hemorrhage, vital sign parameters did not change during over-resuscitation. The strongest linear correlation was observed with pulmonary capillary wedge pressure in both hemorrhage (r2 = 0.99) and volume overload (r2 = 0.98). CONCLUSIONS: Pulmonary capillary wedge pressure is the most accurate parameter to track both hemorrhage and over-resuscitation, demonstrating the unmet clinical need for a less invasive pulmonary capillary wedge pressure equivalent.

Non-Invasive Venous waveform Analysis (NIVA) for volume assessment during complex cranial vault reconstruction: A proof-of-concept study in children.

BACKGROUND: Non-Invasive Venous waveform Analysis (NIVA) is novel technology that captures and analyzes changes in venous waveforms from a piezoelectric sensor on the wrist for hemodynamic volume assessment. Complex cranial vault reconstruction is performed in children with craniosynostosis and is associated with extensive blood loss, potential life-threatening risks, and significant morbidity. In this preliminary study, we hypothesized that NIVA will provide a reliable, non-invasive, quantitative assessment of intravascular volume changes in children undergoing complex cranial vault reconstruction. OBJECTIVE: To present proof-of-concept results of a novel technology in the pediatric population. METHODS: The NIVA prototype was placed on each subject's wrist, and venous waveforms were collected intraoperatively. Estimated blood loss and fluid/blood product administration were recorded in real time. Venous waveforms were analyzed into a NIVA value and then correlated, along with mean arterial pressure (MAP), to volume changes. Concordance was quantified to determine if the direction of change in volume was similar to the direction of change in MAP or change in NIVA. RESULTS: Of 18 patients enrolled, 14 had usable venous waveforms, and there was a significant correlation between change in NIVA value and change in volume. Change in MAP did not correlate with change in volume. The concordance between change in MAP and change in volume was less than the concordance between change in NIVA and change in volume. CONCLUSION: NIVA values correlate more closely to intravascular volume changes in pediatric craniofacial patients than MAP. This initial study suggests that NIVA is a potential safe, reliable, non-invasive quantitative method of measuring intravascular volume changes for children undergoing surgery.

Cell-free hemoglobin increases inflammation, lung apoptosis, and microvascular permeability in murine polymicrobial sepsis.

Increased endothelial permeability is central to the pathogenesis of sepsis and leads to organ dysfunction and death but the endogenous mechanisms that drive increased endothelial permeability are not completely understood. We previously reported that cell-free hemoglobin (CFH), elevated in 80% of patients with sepsis, increases lung microvascular permeability in an ex vivo human lung model and cultured endothelial cells. In this study, we augmented a murine model of polymicrobial sepsis with elevated circulating CFH to test the hypothesis that CFH increases microvascular endothelial permeability by inducing endothelial apoptosis. Mice were treated with an intraperitoneal injection of cecal slurry with or without a single intravenous injection of CFH. Severity of illness, mortality, systemic and lung inflammation, endothelial injury and dysfunction and lung apoptosis were measured at selected time points. We found that CFH added to CS increased sepsis mortality, plasma inflammatory cytokines as well as lung apoptosis, edema and inflammation without affecting large vessel reactivity or vascular injury marker concentrations. These results suggest that CFH is an endogenous mediator of increased endothelial permeability and apoptosis in sepsis and may be a promising therapeutic target.

Observational Study of Noninvasive Venous Waveform Analysis to Assess Intracardiac Filling Pressures During Right Heart Catheterization.

BACKGROUND: Outpatient monitoring and management of patients with heart failure (HF) reduces hospitalizations and health care costs. However, the availability of noninvasive approaches to assess congestion is limited. Noninvasive venous waveform analysis (NIVA) uses a unique physiologic signal, the morphology of the venous waveform, to assess intracardiac filling pressures. This study is a proof of concept analysis of the correlation between NIVA value and pulmonary capillary wedge pressure (PCWP) and the ability of the NIVA value to predict PCWP > 18 mmHg in subjects undergoing elective right heart catheterization (RHC). PCWP was also compared across common clinical correlates of congestion. METHODS AND RESULTS: A prototype NIVA device, which consists of a piezoelectric sensor placed over the skin on the volar aspect of the wrist, connected to a data-capture control box, was used to collect venous waveforms in 96 patients during RHC. PCWP was collected at end-expiration by an experienced cardiologist. The venous waveform signal was transformed to the frequency domain (Fourier transform), where a ratiometric algorithm of the frequencies of the pulse rate and its harmonics was used to derive a NIVA value. NIVA values were successfully captured in 83 of 96 enrolled patients. PCWP ranged from 4-40 mmHg with a median of 13 mmHg. NIVA values demonstrated a linear correlation with PCWP (r = 0.69, P < 0.05). CONCLUSIONS: This observational proof-of-concept study using a prototype NIVA device demonstrates a moderate correlation between NIVA value and PCWP in patients undergoing RHC. NIVA, thus, represents a promising developing technology for noninvasive assessment of congestion in spontaneously breathing patients.

An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles.

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Normal Saline solutions cause endothelial dysfunction through loss of membrane integrity, ATP release, and inflammatory responses mediated by P2X7R/p38 MAPK/MK2 signaling pathways.

Resuscitation with 0.9% Normal Saline (NS), a non-buffered acidic solution, leads to increased morbidity and mortality in the critically ill. The goal of this study was to determine the molecular mechanisms of endothelial injury after exposure to NS. The hypothesis of this investigation is that exposure of endothelium to NS would lead to loss of cell membrane integrity, resulting in release of ATP, activation of the purinergic receptor (P2X7R), and subsequent activation of stress activated signaling pathways and inflammation. Human saphenous vein endothelial cells (HSVEC) incubated in NS, but not buffered electrolyte solution (Plasma-Lyte, PL), exhibited abnormal morphology and increased release of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and decreased transendothelial resistance (TEER), suggesting loss of membrane integrity. Incubation of intact rat aorta (RA) or human saphenous vein in NS but not PL led to impaired endothelial-dependent relaxation which was ameliorated by apyrase (hydrolyzes ATP) or SB203580 (p38 MAPK inhibitor). Exposure of HSVEC to NS but not PL led to activation of p38 MAPK and its downstream substrate, MAPKAP kinase 2 (MK2). Treatment of HSVEC with exogenous ATP led to interleukin 1ß (IL-1ß) release and increased vascular cell adhesion molecule (VCAM) expression. Treatment of RA with IL-1ß led to impaired endothelial relaxation. IL-1ß treatment of HSVEC led to increases in p38 MAPK and MK2 phosphorylation, and increased levels of arginase II. Incubation of porcine saphenous vein (PSV) in PL with pH adjusted to 6.0 or less also led to impaired endothelial function, suggesting that the acidic nature of NS is what contributes to endothelial dysfunction. Volume overload resuscitation in a porcine model after hemorrhage with NS, but not PL, led to acidosis and impaired endothelial function. These data suggest that endothelial dysfunction caused by exposure to acidic, non-buffered NS is associated with loss of membrane integrity, release of ATP, and is modulated by P2X7R-mediated inflammatory responses.

Nanotechnology Enabled Modulation of Signaling Pathways Affects Physiologic Responses in Intact Vascular Tissue.

IMPACT STATEMENT: Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to traditional vasodilators, and inhibition of vasospasm after SAH remains a large unmet clinical need. SAH causes changes in the phosphorylation state of the small heat shock proteins (HSPs), HSP20 and HSP27, in the vasospastic vessels. In this study, the levels of HSP27 and HSP20 were manipulated using nanotechnology to mimic the intracellular phenotype of SAH-induced vasospasm, and the effect of this manipulation was tested on vasomotor responses in intact tissues. This work provides insight into potential therapeutic targets for the development of more effective treatments for SAH induced vasospasm.

Excipients for the lyoprotection of MAPKAP kinase 2 inhibitory peptide nano-polyplexes.

Herein, excipients are investigated to ameliorate the deleterious effects of lyophilization on peptide-polymer nano-polyplex (NP) morphology, cellular uptake, and bioactivity. The NPs are a previously-described platform technology for intracellular peptide delivery and are formulated from a cationic therapeutic peptide and the anionic, pH-responsive, endosomolytic polymer poly(propylacrylic acid) (PPAA). These NPs are effective when formulated and immediately used for delivery into cells and tissue, but they are not amenable to reconstitution following storage as a lyophilized powder due to aggregation. To develop a lyophilized NP format that facilitates longer-term storage and ease of use, MAPKAP kinase 2 inhibitory peptide-based NPs (MK2i-NPs) were prepared in the presence of a range of concentrations of the excipients sucrose, trehalose, and lactosucrose prior to lyophilization and storage. All excipients improved particle morphology post-lyophilization and significantly improved MK2i-NP uptake in human coronary artery smooth muscle cells relative to lyophilized NPs without excipient. In particular, MK2i-NPs lyophilized with 300 mM lactosucrose as an excipient demonstrated a 5.23 fold increase in cellular uptake (p < 0.001), a 2.52 fold increase in endosomal disruption (p < 0.05), and a 2.39 fold increase in ex vivo bioactivity (p < 0.01) compared to MK2i-NPs lyophilized without excipients. In sum, these data suggest that addition of excipients, particularly lactosucrose, maintains and even improves the uptake and therapeutic efficacy of peptide-polymer NPs post-lyophilization relative to freshly-made formulations. Thus, the use of excipients as lyoprotectants is a promising approach for the long-term storage of biotherapeutic NPs and poises this NP platform for clinical translation.

Vascular surgical stretch injury leads to activation of P2X7 receptors and impaired endothelial function.

A viable vascular endothelial layer prevents vasomotor dysfunction, thrombosis, inflammation, and intimal hyperplasia. Injury to the endothelium occurs during harvest and "back table" preparation of human saphenous vein prior to implantation as an arterial bypass conduit. A subfailure overstretch model of rat aorta was used to show that subfailure stretch injury of vascular tissue leads to impaired endothelial-dependent relaxation. Stretch-induced impaired relaxation was mitigated by treatment with purinergic P2X7 receptor (P2X7R) inhibitors, brilliant blue FCF (FCF) and A740003, or apyrase, an enzyme that catalyzes the hydrolysis of ATP. Alternatively, treatment of rat aorta with exogenous ATP or 2'(3')-O-(4-Benzoyl benzoyl)-ATP (BzATP) also impaired endothelial-dependent relaxation. Treatment of human saphenous vein endothelial cells (HSVEC) with exogenous ATP led to reduced nitric oxide production which was associated with increased phosphorylation of the stress activated protein kinase, p38 MAPK. ATP- stimulated p38 MAPK phosphorylation of HSVEC was inhibited by FCF and SB203580. Moreover, ATP inhibition of nitric oxide production in HSVEC was prevented by FCF, SB203580, L-arginine supplementation and arginase inhibition. Finally, L-arginine supplementation and arginase inhibition restored endothelial dependent relaxation after stretch injury of rat aorta. These results suggest that vascular stretch injury leads to ATP release, activation of P2X7R and p38 MAPK resulting in endothelial dysfunction due to arginase activation. Endothelial function can be restored in both ATP treated HSVEC and intact stretch injured rat aorta by P2X7 receptor inhibition with FCF or L-arginine supplementation, implicating straightforward therapeutic options for treatment of surgical vascular injury.

Heparin-functionalized polymer graft surface eluting MK2 inhibitory peptide to improve hemocompatibility and anti-neointimal activity.

The leading cause of synthetic graft failure includes thrombotic occlusion and intimal hyperplasia at the site of vascular anastomosis. Herein, we report a co-immobilization strategy of heparin and potent anti-neointimal drug (Mitogen Activated Protein Kinase II inhibitory peptide; MK2i) by using a tyrosinase-catalyzed oxidative reaction for preventing thrombotic occlusion and neointimal formation of synthetic vascular grafts. The binding of heparin-tyramine polymer (HT) onto the polycarprolactone (PCL) surface enhanced blood compatibility with significantly reduced protein absorption (64.7% decrease) and platelet adhesion (85.6% decrease) compared to bare PCL surface. When loading MK2i, 1) the HT depot surface gained high MK2i-loading efficiency through charge-charge interaction, and 2) this depot platform enabled long-term, controlled release over 4weeks (92-272µg/mL of MK2i). The released MK2i showed significant inhibitory effects on VSMC migration through down-regulated phosphorylation of target proteins (HSP27 and CREB) associated with intimal hyperplasia. In addition, it was found that the released MK2i infiltrated into the tissue with a cumulative manner in ex vivo human saphenous vein (HSV) model. This present study demonstrates that enzymatically HT-coated surface modification is an effective strategy to induce long-term MK2i release as well as hemocompatibility, thereby improving anti-neointimal activity of synthetic vascular grafts.

Limiting Injury During Saphenous Vein Graft Preparation For Coronary Arterial Bypass Prevents Metabolic Decompensation.

Standard harvest and preparation of human saphenous vein (HSV) for autologous coronary and peripheral arterial bypass procedures is associated with injury and increased oxidative stress that negatively affect graft performance. In this study we investigated the global metabolomic profiles of HSV before (unprepared; UP) and after standard vein graft preparation (AP). AP-HSV showed impaired vasomotor function that was associated with increased oxidative stress, phospholipid hydrolysis and energy depletion that are characteristic of mechanical and chemical injury. A porcine model (PSV) was utilized to validate these metabolomic changes in HSV and to determine the efficacy of an improved preparation technique (OP) using pressure-regulated distension, a non-toxic vein marker, and graft storage in buffered PlasmaLyte solution in limiting metabolic decompensation due to graft preparation. Deficits in vasomotor function and metabolic signature observed in AP-PSV could be largely mitigated with the OP procedure. These findings suggest that simple strategies aimed at reducing injury during graft harvest and preparation represents a straightforward and viable strategy to preserve conduit function and possibly improve graft patency.

Adenosine triphosphate as a molecular mediator of the vascular response to injury.

BACKGROUND: Human saphenous veins used for arterial bypass undergo stretch injury at the time of harvest and preimplant preparation. Vascular injury promotes intimal hyperplasia, the leading cause of graft failure, but the molecular events leading to this response are largely unknown. This study investigated adenosine triphosphate (ATP) as a potential molecular mediator in the vascular response to stretch injury, and the downstream effects of the purinergic receptor, P2X7R, and p38 MAPK activation. MATERIALS AND METHODS: A subfailure stretch rat aorta model was used to determine the effect of stretch injury on release of ATP and vasomotor responses. Stretch-injured tissues were treated with apyrase, the P2X7R antagonist, A438079, or the p38 MAPK inhibitor, SB203580, and subsequent contractile forces were measured using a muscle bath. An exogenous ATP (eATP) injury model was developed and the experiment repeated. Change in p38 MAPK phosphorylation after stretch and eATP tissue injury was determined using Western blotting. Noninjured tissue was incubated in the p38 MAPK activator, anisomycin, and subsequent contractile function and p38 MAPK phosphorylation were analyzed. RESULTS: Stretch injury was associated with release of ATP. Contractile function was decreased in tissue subjected to subfailure stretch, eATP, and anisomycin. Contractile function was restored by apyrase, P2X7R antagonism, and p38-MAPK inhibition. Stretch, eATP, and anisomycin-injured tissue demonstrated increased phosphorylation of p38 MAPK. CONCLUSIONS: Taken together, these data suggest that the vascular response to stretch injury is associated with release of ATP and activation of the P2X7R/P38 MAPK pathway, resulting in contractile dysfunction. Modulation of this pathway in vein grafts after harvest and before implantation may reduce the vascular response to injury.

Unregulated saphenous vein graft distension decreases tissue viscoelasticity.

OBJECTIVES: Unregulated intraoperative distension of human saphenous vein (SV) graft leads to supraphysiologic luminal pressures and causes acute physiologic and cellular injury to the conduit. The effect of distension on tissue viscoelasticity, a biophysical property critical to a successful graft, is not well described. In this investigation, we quantify the loss of viscoelasticity in SV deformed by distension and compare the results to tissue distended in a pressure-controlled fashion. MATERIALS AND METHODS: Unmanipulated porcine SV was used as a control or distended without regulation and distended with an in-line pressure release valve (PRV). Rings were cut from these tissues and suspended on a muscle bath. Force versus time tracings of tissue constricted with KCl (110 mM) and relaxed with sodium nitroprusside (SNP) were fit to the Hill model of viscoelasticity, using mean absolute error (MAE) and r2-goodness of fit as measures of conformity. RESULTS: One-way ANOVA analysis demonstrated that, in tissue distended manually, the MAE was significantly greater and the r2-goodness of fit was significantly lower than both undistended tissues and tissues distended with a PRV (p<0.05) in KCl-induced vasoconstriction and SNP-induced vasodilation. CONCLUSIONS: Unregulated manual distension of SV graft causes loss of viscoelasticity and such loss may be mitigated with the use of an in-line PRV.

Upper Extremity Pulse Pressure Predicts Amputation-Free Survival after Lower Extremity Bypass.

Increased pulse pressure reflects pathologic arterial stiffening and predicts cardiovascular events and mortality. The effect of pulse pressure on outcomes in lower extremity bypass patients remains unknown. We thus investigated whether preoperative pulse pressure could predict amputation-free survival in patients undergoing lower extremity bypass for atherosclerotic occlusive disease. An institutional database identified 240 included patients undergoing lower extremity bypass from 2005 to 2014. Preoperative demographics, cardiovascular risk factors, operative factors, and systolic and diastolic blood pressures were recorded, and compared between patients with pulse pressures above and below 80 mm Hg. Factors were analyzed in bi- and multivariable models to assess independent predictors of amputation-free survival. Kaplan-Meier analysis was performed to evaluate the temporal effect of pulse pressure ≥80 mm Hg on amputation-free survival. Patients with a pulse pressure ≥80 mm Hg were older, male, and had higher systolic and lower diastolic pressures. Patients with pulse pressure <80 mm Hg demonstrated a survival advantage on Kaplan-Meier analysis at six months (log-rank P = 0.003) and one year (P = 0.005) postoperatively. In multivariable analysis, independent risk factors for decreased amputation-free survival at six months included nonwhite race, tissue loss, infrapopliteal target, and preoperative pulse pressure ≥80 mm Hg (hazard ratio 2.60; P = 0.02), while only tissue loss and pulse pressure ≥80 mm Hg (hazard ratio 2.30, P = 0.02) remained predictive at one year. Increased pulse pressure is independently associated with decreased amputation-free survival in patients undergoing lower extremity bypass. Further efforts to understand the relationship between increased arterial stiffness and poor outcomes in these patients are needed.

Prediction of Prolonged Ventilation after Coronary Artery Bypass Grafting: Data from an Artificial Neural Network.

OBJECTIVES: The need for mechanical ventilation 24 hours after coronary artery bypass grafting (CABG) is considered a morbidity by the Society of Thoracic Surgeons. The purpose of this investigation was twofold: to identify simple preoperative patient factors independently associated with prolonged ventilation and to optimize prediction and early identification of patients prone to prolonged ventilation using an artificial neural network (ANN). METHODS: Using the institutional Adult Cardiac Database, 738 patients who underwent CABG since 2005 were reviewed for preoperative factors independently associated with prolonged postoperative ventilation. Prediction of prolonged ventilation from the identified variables was modeled using both "traditional" multiple logistic regression and an ANN. The two models were compared using Pearson r2 and area under the curve (AUC) parameters. RESULTS: Of 738 included patients, 14% (104/738) required mechanical ventilation ≥ 24 hours postoperatively. Upon multivariate analysis, higher body-mass index (BMI; odds ratio [OR] 1.10 per unit, P < 0.001), lower ejection fraction (OR 0.97 per %, P = 0.01) and use of cardiopulmonary bypass (OR 2.59, P = 0.02) were independently predictive of prolonged ventilation. The Pearson r2 and AUC of the multivariate nominal logistic regression model were 0.086 and 0.698 ± 0.05, respectively; analogous statistics of the ANN model were 0.159 and 0.732 ± 0.05, respectively.BMI, ejection fraction and cardiopulmonary bypass represent three simple factors that may predict prolonged ventilation after CABG. Early identification of these patients can be optimized using an ANN, an emerging paradigm for clinical outcomes modeling that may consider complex relationships among these variables.

Calcification of Human Saphenous Vein Associated with Endothelial Dysfunction: A Pilot Histopathophysiological and Demographical Study.

While the pathophysiology and clinical significance of arterial calcifications have been studied extensively, minimal focus has been placed on venous calcification deposition. In this study, we evaluated the association between calcium deposition in human saphenous vein (HSV), endothelial function, and patient demographic risk factors. Fifty-four HSV segments were collected at the time of coronary artery bypass graft (CABG) surgery. The presence or absence of calcium deposits was visualized using the Von Kossa staining method. Endothelial function was determined by measuring muscle tissue contraction with phenylephrine and relaxation with carbachol in a muscle bath. Additional segments of vein underwent histologic evaluation for preexisting intimal thickness and extracellular matrix (ECM) deposition. Patient demographics data were obtained through our institution's electronic medical record, with patient consent. Calcium was present in 16 of 54 samples (29.6%). Veins with calcium deposits had significantly greater intimal-to-medial thickness ratios (p = 0.0058) and increased extracellular collagen deposition (p = 0.0077). Endothelial relaxation was significantly compromised in calcified veins vs. those without calcium (p = 0.0011). Significant patient risk factors included age (p = 0.001) and preoperative serum creatinine (p = 0.017). Calcified veins can be characterized as having endothelial dysfunction with increased basal intimal thickness and increased ECM deposition. Patient risk factors for calcium deposits in veins were similar to those for arteries, namely, advanced age and kidney disease. Further studies are needed to determine the effect of preexisting vein calcification on short- and long-term graft patency.