A cell permeant phosphopeptide mimetic of Niban inhibits p38 MAPK and restores endothelial function after injury.

Vascular injury leads to membrane disruption, ATP release, and endothelial dysfunction. Increases in the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and decreases in the phosphorylation of Niban, a protein implicated in ER stress and apoptosis, are associated with vascular injury. A cell permeant phosphopeptide mimetic of Niban (NiPp) was generated. The effects of NiPp in restoring endothelial function were determined ex vivo using intact rat aortic tissue (RA) after pharmacological activation of p38 MAPK and also in multiple clinically relevant injury models. Anisomycin (Aniso) increased p38 MAPK phosphorylation and reduced endothelial-dependent relaxation in RA. Treatment with NiPp prevented Ansio-induced reduction in endothelial function and increases in p38 MAPK phosphorylation. NiPp treatment also restored endothelial function after stretch injury (subfailure stretch), treatment with acidic Normal Saline (NS), and P2X7R activation with 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Aged, diseased, human saphenous vein (HSV) remnants obtained from patients undergoing coronary bypass surgical procedures have impaired endothelial function. Treatment of these HSV segments with NiPp improved endothelial-dependent relaxation. Kinome screening experiments indicated that NiPp inhibits p38 MAPK. These data demonstrate that p38 MAPK and Niban signaling have a role in endothelial function, particularly in response to injury. Niban may represent an endogenous regulator of p38 MAPK activation. The NiPp peptide may serve as an experimental tool to further elucidate p38 MAPK regulation and as a potential therapeutic for endothelial dysfunction.

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.

P2X7R antagonism after subfailure overstretch injury of blood vessels reverses vasomotor dysfunction and prevents apoptosis.

Human saphenous vein (HSV) is harvested and prepared prior to implantation as an arterial bypass graft. Injury and the response to injury from surgical harvest and preparation trigger cascades of molecular events and contribute to graft remodeling and intimal hyperplasia. Apoptosis is an early response after implantation that contributes the development of neointimal lesions. Here, we showed that surgical harvest and preparation of HSV leads to vasomotor dysfunction, increased apoptosis and downregulation of the phosphorylation of the anti-apoptotic protein, Niban. A model of subfailure overstretch injury in rat aorta (RA) was used to demonstrate impaired vasomotor function, increased extracellular ATP (eATP) release, and increased apoptosis following pathological vascular injury. The subfailure overstretch injury was associated with activation of p38 MAPK stress pathway and decreases in the phosphorylation of the anti-apoptotic protein Niban. Treatment of RA after overstretch injury with antagonists to purinergic P2X7 receptor (P2X7R) antagonists or P2X7R/pannexin (PanX1) complex, but not PanX1 alone, restored vasomotor function. Inhibitors to P2X7R and PanX1 reduced stretch-induced eATP release. P2X7R/PanX1 antagonism led to decrease in p38 MAPK phosphorylation, restoration of Niban phosphorylation and increases in the phosphorylation of the anti-apoptotic protein Akt in RA and reduced TNFα-stimulated caspase 3/7 activity in cultured rat vascular smooth muscle cells. In conclusion, inhibition of P2X7R after overstretch injury restored vasomotor function and inhibited apoptosis. Treatment with P2X7R/PanX1 complex inhibitors after harvest and preparation injury of blood vessels used for bypass conduits may prevent the subsequent response to injury that lead to apoptosis and represents a novel therapeutic approach to prevent graft failure.

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.

Heat Shock-Related Protein 20 Peptide Decreases Human Airway Constriction Downstream of ß2-Adrenergic Receptor.

Severe bronchospasm refractory to ß-agonists is a challenging aspect of asthma therapy, and novel therapeutics are needed. ß-agonist-induced airway smooth muscle (ASM) relaxation is associated with increases in the phosphorylation of the small heat shock-related protein (HSP) 20. We hypothesized that a transducible phosphopeptide mimetic of HSP20 (P20 peptide) causes relaxation of human ASM (HASM) by interacting with target(s) downstream of the ß2-adrenergic receptor (ß2AR) pathway. The effect of the P20 peptide on ASM contractility was determined in human and porcine ASM using a muscle bath. The effect of the P20 peptide on filamentous actin dynamics and migration was examined in intact porcine ASM and cultured primary HASM cells. The efficacy of the P20 peptide in vivo on airway hyperresponsiveness (AHR) was determined in an ovalbumin (OVA) sensitization and challenge murine model of allergic airway inflammation. P20 peptide caused dose-dependent relaxation of carbachol-precontracted ASM and blocked carbachol-induced contraction. The ß2AR inhibitor, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551), abrogated isoproterenol but not P20 peptide-mediated relaxation. The P20 peptide decreased filamentous actin levels in intact ASM, disrupted stress fibers, and inhibited platelet-derived growth factor-induced migration of HASM cells. The P20 peptide treatment reduced methacholine-induced AHR in OVA mice without affecting the inflammatory response. These results suggest that the P20 peptide decreased airway constriction and disrupted stress fibers through regulation of the actin cytoskeleton downstream of ß2AR. Thus, the P20 peptide may be a potential therapeutic for asthma refractory to ß-agonists.

Use of Brilliant Blue FCF during vein graft preparation inhibits intimal hyperplasia.

BACKGROUND: Intimal hyperplasia remains the primary cause of vein graft failure for the 1 million yearly bypass procedures performed using human saphenous vein (HSV) grafts. This response to injury is caused in part by the harvest and preparation of the conduit. The use of Brilliant Blue FCF (FCF) restores injury-induced loss of function in vascular tissues possibly via inhibition of purinergic receptor signaling. This study investigated whether pretreatment of the vein graft with FCF prevents intimal hyperplasia. METHODS: Cultured rat aortic smooth muscle cells (A7r5) were used to determine the effect of FCF on platelet-derived growth factor-mediated migration and proliferation, cellular processes that contribute to intimal hyperplasia. The effectiveness of FCF treatment during the time of explantation on preventing intimal hyperplasia was evaluated in a rabbit jugular-carotid interposition model and in an organ culture model using HSV. RESULTS: FCF inhibited platelet-derived growth factor-induced migration and proliferation of A7r5 cells. Treatment with FCF at the time of vein graft explantation inhibited the subsequent development of intimal thickening in the rabbit model. Pretreatment with FCF also prevented intimal thickening of HSV in organ culture. CONCLUSIONS: Incorporation of FCF as a component of vein graft preparation at the time of explantation represents a potential therapeutic approach to mitigate intimal hyperplasia, reduce vein graft failure, and improve outcome of the autologous transplantation of HSV.

Brilliant blue FCF is a nontoxic dye for saphenous vein graft marking that abrogates response to injury.

BACKGROUND: Injury to saphenous vein grafts during surgical preparation may contribute to the subsequent development of intimal hyperplasia, the primary cause of graft failure. Surgical skin markers currently used for vascular marking contain gentian violet and isopropanol, which damage tissue and impair physiologic functions. Brilliant blue FCF (FCF) is a nontoxic dye alternative that may also ameliorate preparation-induced injury. METHODS: Porcine saphenous vein (PSV) was used to evaluate the effect of FCF on physiologic responses in a muscle bath. Cytotoxicity of FCF was measured using human umbilical venous smooth muscle cells. Effect of FCF on the development of intimal hyperplasia was evaluated in organ culture using PSV. Intracellular calcium fluxes and contractile responses were measured in response to agonists and inhibitors in rat aorta and human saphenous vein. RESULTS: Marking with FCF did not impair smooth muscle contractile responses and restored stretch injury-induced loss in smooth muscle contractility of PSV. Gentian violet has cytotoxic effects on human umbilical venous smooth muscle cells, whereas FCF is nontoxic. FCF inhibited intimal thickening in PSV in organ culture. Contraction induced by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate and intracellular calcium flux were inhibited by FCF, oxidized adenosine triphosphate, KN-62, and brilliant blue G, suggesting that FCF may inhibit the purinergic receptor P2X7. CONCLUSIONS: Our studies indicated that FCF is a nontoxic marking dye for vein grafts that ameliorates vein graft injury and prevents intimal thickening, possibly due to P2X7 receptor inhibition. FCF represents a nontoxic alternative for vein graft marking and a potentially therapeutic approach to enhance outcome in autologous transplantation of human saphenous vein into the coronary and peripheral arterial circulation.

Traditional graft preparation decreases physiologic responses, diminishes viscoelasticity, and reduces cellular viability of the conduit: A porcine saphenous vein model.

Traditional methods of intraoperative human saphenous vein preparation for use as bypass grafts can be deleterious to the conduit. The purpose of this study was to characterize acute graft preparation injury, and to mitigate this harm via an improved preparation technique. Porcine saphenous veins were surgically harvested (unprepared controls, UnP) and prepared using traditional (TraP) and improved preparations (ImP). The TraP used unregulated radial distension, marking with a surgical skin marker and preservation in heparinized normal saline. ImP used pressure-regulated distension, brilliant blue FCF-based pen marking and preservation in heparinized Plasma-Lyte A. Rings from each preparation were suspended in a muscle bath for characterization of physiologic responses to vasoactive agents and viscoelasticity. Cellular viability was assessed using the methyl thiazolyl tetrazolium (MTT) assay and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for apoptosis. Contractile responses to potassium chloride (110 mM) and phenylephrine (10 µM), and endothelial-dependent and independent vasodilatory responses to carbachol (0.5 µM) and sodium nitroprusside (1 µM), respectively, were decreased in TraP tissues compared to both UnP and ImP tissues (p ⩽ 0.05). TraP tissues demonstrated diminished viscoelasticity relative to UnP and ImP tissues (p ⩽ 0.05), and reduced cellular viability relative to UnP control (p ⩽ 0.01) by the MTT assay. On the TUNEL assay, TraP tissues demonstrated a greater degree of apoptosis relative to UnP and ImP tissues (p ⩽ 0.01). In conclusion, an improved preparation technique prevents vascular graft smooth muscle and endothelial injury observed in tissues prepared using a traditional approach.

Biomaterial-Based Approaches to Address Vein Graft and Hemodialysis Access Failures.

Veins used as grafts in heart bypass or as access points in hemodialysis exhibit high failure rates, thereby causing significant morbidity and mortality for patients. Interventional or revisional surgeries required to correct these failures have been met with limited success and exorbitant costs, particularly for the US Centers for Medicare & Medicaid Services. Vein stenosis or occlusion leading to failure is primarily the result of neointimal hyperplasia. Systemic therapies have achieved little long-term success, indicating the need for more localized, sustained, biomaterial-based solutions. Numerous studies have demonstrated the ability of external stents to reduce neointimal hyperplasia. However, successful results from animal models have failed to translate to the clinic thus far, and no external stent is currently approved for use in the US to prevent vein graft or hemodialysis access failures. This review discusses current progress in the field, design considerations, and future perspectives for biomaterial-based external stents. More comparative studies iteratively modulating biomaterial and biomaterial-drug approaches are critical in addressing mechanistic knowledge gaps associated with external stent application to the arteriovenous environment. Addressing these gaps will ultimately lead to more viable solutions that prevent vein graft and hemodialysis access failures.

Surgical vein graft preparation promotes cellular dysfunction, oxidative stress, and intimal hyperplasia in human saphenous vein.

INTRODUCTION: Human saphenous vein (HSV) is the most widely used bypass conduit for peripheral and coronary vascular reconstructions. However, outcomes are limited by a high rate of intimal hyperplasia (IH). HSV undergoes a series of ex vivo surgical manipulations prior to implantation, including hydrostatic distension, marking, and warm ischemia in solution. We investigated the impact of surgical preparation on HSV cellular function and development of IH in organ culture. We hypothesized that oxidative stress is a mediator of HSV dysfunction. METHODS: HSV was collected from patients undergoing vascular bypass before and after surgical preparation. Smooth muscle and endothelial function were measured using a muscle bath. Endothelial preservation was assessed with immunohistochemical staining. An organ culture model was used to investigate the influence of surgical preparation injury on the development of IH. Superoxide levels were measured using a high-performance liquid chromatography-based assay. The influence of oxidative stress on HSV physiologic responses was investigated by exposing HSV to hydrogen peroxide (H2O2). RESULTS: Surgical vein graft preparation resulted in smooth muscle and endothelial dysfunction, endothelial denudation, diminished endothelial nitric oxide synthase staining, development of increased IH, and increased levels of reactive oxygen species. Experimental induction of oxidative stress in unmanipulated HSV by treatment with H2O2 promoted endothelial dysfunction. Duration of storage time in solution did not contribute to smooth muscle or endothelial dysfunction. CONCLUSIONS: Surgical vein graft preparation causes dysfunction of the smooth muscle and endothelium, endothelial denudation, reduced endothelial nitric oxide synthase expression, and promotes IH in organ culture. Moreover, increased levels of reactive oxygen species are produced and may promote further vein graft dysfunction. These results argue for less injurious means of preparing HSV prior to autologous transplantation into the arterial circulation.

A Porcine Model of Acute Respiratory Failure with a Continuous Infusion of Oleic Acid.

This protocol outlines an acute respiratory distress model utilizing centrally administered oleic acid infusion in Yorkshire pigs. Prior to experimentation, each pig underwent general anesthesia, endotracheal intubation, and mechanical ventilation, and was equipped with bilateral jugular vein central vascular access catheters. Oleic acid was administered through a dedicated pulmonary artery catheter at a rate of 0.2 mL/kg/h. The infusion lasted for 60-120 min, inducing respiratory distress. Throughout the experiment, various parameters including heart rate, respiratory rate, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, end-tidal carbon dioxide, peak airway pressures, and plateau pressures were monitored. Around the 60 min mark, decreases in partial arterial oxygen pressure (PaO2) and fraction of oxygen-saturated hemoglobin (SpO2) were observed. Periodic hemodynamic instability, accompanied by acute increases in pulmonary artery pressures, occurred during the infusion. Post-infusion, histological analysis of the lung parenchyma revealed changes indicative of parenchymal damage and acute disease processes, confirming the effectiveness of the model in simulating acute respiratory decompensation.

The Cell Permeant Phosphopetpide mimetic of VASP Alleviates Motor Function Deficits After Experimental Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) due to the rupture of an intracranial aneurysm leads to delayed vasospasm and neuroischemia, which can result in profound neurologic deficit and death. Therapeutic options after SAH are currently limited to hemodynamic optimization and nimodipine, which have limited clinical efficacy. Experimental SAH results in cerebral vasospasm have demonstrated the downregulation of nitric oxide (NO)-protein kinase G (PKG) signaling elements. VP3 is a novel cell permeant phosphopeptide mimetic of VASP, a substrate of PKG and an actin-associated protein that modulates vasorelaxation in vascular smooth muscle cells. In this study, we determined that intravenous administration of high doses of VP3 did not induce systemic hypotension in rats except at the maximal soluble dose, implying that VP3 is well-tolerated and has a wide therapeutic window. Using a single cisterna magna injection rat model of SAH, we demonstrated that intravenous administration of low-dose VP3 after SAH improved neurologic deficits for up to 14 days as determined by the rotarod test. These findings suggest that strategies aimed at targeting the cerebral vasculature with VP3 may improve neurologic deficits associated with SAH.

Adult and Pediatric Porcine Model of Acute Volume Overload.

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, endotracheal intubation, and mechanical ventilation. A central venous catheter and an arterial catheter are placed via surgical cutdown in the external jugular vein and carotid artery, respectively. A pulmonary artery catheter is placed through an introducer sheath of the central venous catheter. PlasmaLyte crystalloid solution is then administered at a rate of 100 mL/min in adult pigs and at 20 mL/kg boluses over 10 min in piglets. Hypervolemia is achieved either at 15% decrease in cardiac output or at 5 L in adult pigs and at 500 mL in piglets. Hemodynamic data, such as heart rate, respiratory rate, end-tidal carbon dioxide, fraction of oxygen-saturated hemoglobin, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, partial arterial oxygen pressure, lactate, pH, base excess, and pulmonary artery fraction of oxygen-saturated hemoglobin, are monitored during experimentation. Preliminary data observed with this model has demonstrated statistically significant changes and strong linear regressions between central hemodynamic parameters and acute volume overload in adult pigs. Only pulmonary capillary wedge pressure demonstrated both a linear regression and a statistical significance to acute volume overload in piglets. These models can aid scientists in the discovery of age-appropriate therapeutic and monitoring strategies to understand and prevent acute volume overload.

Intimal thickness associated with endothelial dysfunction in human vein grafts.

BACKGROUND: Intimal hyperplasia is a complex process thought to be initiated by injury and is the leading cause of vein graft failure. In the present investigation, we hypothesized that the basal intimal thickness in the human saphenous vein is a predictor of endothelial dysfunction and, potentially, intimal hyperplasia. METHODS: Human saphenous veins were obtained during coronary artery bypass surgery. The segments were contracted with phenylephrine and relaxed with carbachol to determine the endothelial-dependent relaxation. The vein segments were fixed in 10% buffered formalin and grown for 14 d in high-serum culture and then fixed in formalin. The fixed tissues were stained with Verhoeff-Van Gieson, and the average intimal and medial thicknesses were calculated using light microscopy and a computerized image analysis system. RESULTS: The human saphenous veins displayed varying amounts of basal intimal thickness (range 18.80-241.3 µm). The endothelial-dependent relaxation of the veins was highly variable, with values ranging from 0% to 27.59%. Human saphenous veins with a basal intimal thickness greater than 120 µm had significantly less endothelial-dependent relaxation (8.90% ± 6.32%) than those with a basal intimal thickness less than 120 µm (21.97% ± 10.64%). Endothelial dysfunction correlated with a basal intimal thickness greater than 120 µm (P = 0.02). The basal intimal thickness also correlated with increased intimal thickness after 14 d in organ culture (P = 0.0001). CONCLUSIONS: A basal intimal thickness greater than 120 µm is a predictor of endothelial dysfunction. Also, because a greater basal intimal thickness correlated with an increased intimal thickness after organ culture, the basal intimal thickness might predict vein graft failure owing to intimal hyperplasia.

Role of the renin-angiotensin system in the pathogenesis of intimal hyperplasia: therapeutic potential for prevention of vein graft failure?

The saphenous vein remains the most widely used conduit for peripheral and coronary revascularization despite a high rate of vein graft failure. The most common cause of vein graft failure is intimal hyperplasia. No agents have been proven to be successful for the prevention of intimal hyperplasia in human subjects. The renin-angiotensin system is essential in the regulation of vascular tone and blood pressure in physiologic conditions. However, this system mediates cardiovascular remodeling in pathophysiologic states. Angiotensin II is becoming increasingly recognized as a potential mediator of intimal hyperplasia. Drugs modulating the renin-angiotensin system include angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. These drugs are powerful inhibitors of atherosclerosis and cardiovascular remodeling, and they are first-line agents for management of several medical conditions based on class I evidence that they delay progression of cardiovascular disease and improve survival. Several experimental models have demonstrated that these agents are capable of inhibiting intimal hyperplasia. However, there are no data supporting their role in prevention of intimal hyperplasia in patients with vein grafts. This review summarizes the physiology of the renin-angiotensin system, the role of angiotensin II in the pathogenesis of cardiovascular remodeling, the medical indications for these agents, and the experimental data supporting an important role of the renin-angiotensin system in the pathogenesis of intimal hyperplasia.

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.

Detrimental effects of mechanical stretch on smooth muscle function in saphenous veins.

OBJECTIVE: This study evaluated the smooth muscle functional response and viability of human saphenous vein (HSV) grafts after harvest and explored the effect of mechanical stretch on contractile responses of porcine saphenous vein (PSV). METHODS: The contractile responses (stress, 10(5) N/m(2)) of deidentified, remnant HSV grafts to depolarizing potassium chloride and the agonist norepinephrine were measured in a muscle organ bath. Cellular viability was evaluated using a methyl thiazole tetrazolium (MTT) assay. A PSV model was used to evaluate the effect of radial, longitudinal, and angular stretch on smooth muscle contractile responses. RESULTS: Contractile responses varied greatly in HSV harvested for autologous vascular and coronary bypass procedures (0.04198 ± 0.008128 × 10(5) N/m(2) to 0.1192 ± 0.02776 × 10(5) N/m(2)). Contractility of the HSV correlated with the cellular viability of the grafts. In the PSV model, manual radial distension of ≥ 300 mm Hg had no impact on the smooth muscle responses of PSV to potassium chloride. Longitudinal and angular stretch significantly decreased the contractile function of PSV by 33.16% and 15.26%, respectively (P < .03). CONCLUSIONS: There is considerable variability in HSV harvested for use as an autologous conduit. Longitudinal and angular stretching during surgical harvest impairs contractile responsiveness of the smooth muscle in saphenous vein. Avoiding stretch-induced injuries to the conduits during harvest and preparation for implantation may reduce adverse biologic responses in the graft (eg, intimal hyperplasia) and improve patency of autologous vein graft bypasses.

Vasorelaxing cell permeant phosphopeptide mimetics for subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm leads to vasospasm resulting in delayed cerebral ischemia. Therapeutic options are currently limited to hemodynamic optimization and nimodipine, which have marginal clinical efficacy. Nitric oxide (NO) modulates cerebral blood flow through activation of the cGMP-Protein Kinase G (PKG) pathway. Our hypothesis is that SAH results in downregulation of signaling components in the NO-PKG pathway which could explain why treatments for vasospasm targeting this pathway lack efficacy and that treatment with a cell permeant phosphopeptide mimetic of downstream effector prevents delayed vasospasm after SAH. Using a rat endovascular perforation model, reduced levels of NO-PKG pathway molecules were confirmed. Additionally, it was determined that expression and phosphorylation of a PKG substrate: Vasodilator-stimulated phosphoprotein (VASP) was downregulated. A family of cell permeant phosphomimetic of VASP (VP) was wasdesigned and shown to have vasorelaxing property that is synergistic with nimodipine in intact vascular tissuesex vivo. Hence, treatment targeting the downstream effector of the NO signaling pathway, VASP, may bypass receptors and signaling elements leading to vasorelaxation and that treatment with VP can be explored as a therapeutic strategy for SAH induced vasospasm and ameliorate neurological deficits.

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.

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.