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Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to pharmacologic intervention. SAH vasospasm is associated with decreased phosphorylation of intracellular heat shock protein (HSP)20 and increased phosphorylation of HSP27. Phosphorylated HSP20 (p-HSP20) is associated with vascular smooth muscle relaxation and phosphorylated HSP27 with contraction and impaired relaxation. This study was undertaken to modulate these key signaling molecules using intracellular delivery technologies to elucidate the effect of modulation on vasomotor tone of intact vascular tissues. Rat aorta was used as a model system to elucidate the impact of HSP20/HSP27 modulation on physiologic vasomotor responses. HSP20 expression was silenced through siRNA transfection utilizing an endosomolytic diblock copolymer. The diblock copolymer self-assembles into small (∼50 nm) serum stable micellar nanoparticles that effectively package siRNA and facilitate tissue penetration, cell uptake, endosomal escape, intracellular bioavailability, and tissue-level silencing of target gene expression. To increase HSP27 activity, recombinant HSP27 was fused to a cell permeant peptide domain to facilitate protein uptake and activity. Furthermore, a cell permeant peptide mimetic of p-HSP20 was delivered through formulation into nano-polyplexes using a pH-responsive endosomolytic polymer. The impact of these biomolecular modulations on physiologic vasomotor responses in intact tissue was then quantified in a muscle bath. Treatment of rat aorta with HSP20 siRNA polymeric nanocarriers decreased expression of HSP20 which was associated with enhanced contractile responses to phenylephrine (PE) and impaired relaxation responses to sodium nitroprusside. Delivery of recombinant HSP27 to the tissue was associated with increased contractile responses to PE. Treatment with a peptide mimetic of p-HSP20 resulted in decreased contractile responses to PE. These results demonstrate that manipulation of protein levels of the small heat shock phosphoproteins, HSP20 and HSP27, in intact vascular tissues is associated with changes in vasomotor responses. In particular, decreasing HSP20 expression and addition of exogenous HSP27 led to enhanced contractility and impaired relaxation. This biochemical signature is similar to the phenotype of SAH associated vasospasm and suggests that alterations in downstream signaling events may be responsible for SAH induced vasospasm being refractory to upstream, receptor-mediated pharmacologic interventions. 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.

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.

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.

Severe bronchospasm refractory to β-agonists is a challenging aspect of asthma therapy, and novel therapeutics are needed. β-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 β2-adrenergic receptor (β2AR) 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 β2AR 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 β2AR. Thus, the P20 peptide may be a potential therapeutic for asthma refractory to β-agonists.

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 P2X 7 . 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 P2X 7 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.

Recent clinical data suggest intraoperative preservation of human saphenous vein (HSV) in normal saline is associated with vein graft failure. We evaluated the influence of several preservation media on acute physiologic function and cellular viability of HSV conduit.Unprepared (UP) HSV obtained from coronary artery bypass graft patients was characterized on a muscle bath after 2-hour storage in 6 solutions: Plasma-Lyte A, 0.9% NaCl (normal saline), University of Wisconsin solution, Celsior solution, autologous whole blood, or glutathione-ascorbic acid L-arginine (GALA) solution. Vascular smooth muscle contractility was assessed after exposure to depolarizing KCl and phenylephrine. The relaxation of phenylephrine-precontracted HSV to sodium nitroprusside and carbachol (endothelial-independent and -dependent relaxation, respectively) was also assessed. Cellular viability was determined via the methyl thiazolyl tetrazolium (MTT) assay. Rat aortae were used to assess the effect of pH during graft preservation on endothelial-dependent relaxation.Preservation of HSV in normal saline and autologous whole blood impaired contractile responses to KCl relative to UP tissues, whereas preservation in University of Wisconsin solution and Celsior solution enhanced contractile responses (P.05). Relative to UP tissues, responses to phenylephrine were decreased with preservation in normal saline, whereas preservation in University of Wisconsin solution, Celsior solution, and GALA all potentiated these responses (P.05). Only preservation in normal saline impaired endothelial-independent relaxation (P = .005). Preservation in Plasma-Lyte A (P = .02), normal saline (P = .002), and University of Wisconsin solution (P = .02) impaired endothelial-dependent relaxation. Normal saline preservation decreased MTT viability index relative to UP tissues (0.02 ± 0.002 mg(-1)0.5 mL(-1) vs 0.033 ± 0.005 mg(-1)0.5 mL(-1); P = .03). Endothelial function was impaired by acidic pH in rat aorta.Preservation of HSV in normal saline causes graft injury leading to impaired physiologic function and decreased viability of the HSV. This harm is mitigated by the use of buffered salt solutions as preservation media.

IntroductionHuman 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.MethodsHSV 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).ResultsSurgical 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.ConclusionsSurgical 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.Clinical RelevanceApproximately 1,000,000 aortocoronary and peripheral vascular reconstructions are performed annually using human saphenous vein grafts. However, outcomes from this procedure are limited by high rates of graft failure. The leading cause of vein graft failure is intimal hyperplasia. A multifactorial process, intimal hyperplasia is thought to arise at least in part due to vein graft injury. Significant trauma occurs to the graft during surgical harvest and subsequent preparation, significantly impairing cellular function and increasing oxidative stress. Efforts to reduce early vein graft injury during harvest and preparation may have the potential to reduce subsequent vein graft failure in patients.

Human saphenous vein (HSV) remains the most widely used conduit for peripheral and coronary bypass procedures. However, data from the Project of Ex-Vivo vein graft Engineering via Transfection-III (PREVENT III) trial revealed vein graft failure rates of HSV as high as 40% at one year postoperatively.(1) While early vein graft failure (3,000 patients who underwent coronary artery bypass grafting) was stratified by type of preservation solution, including normal saline, buffered salt solutions or autologous whole blood. Controlling for baseline demographics and operative characteristics, those patients for whom a buffered salt solution (e.g. Plasma-Lyte A, University of Wisconsin solution) was used for graft preservation had a significant improvement in vein graft failure at one year, and trended toward a significant benefit at five years. Balanced salt solutions, designed considering physiologic pH and electrolyte composition, better preserved the HSV graft and reduced vein graft failure.(4) HSV graft is still frequently preserved in normal saline solution prior to implantation. Normal saline, while inexpensive and readily available, is acidic, with a pH < 6.0, mildly hyperosmolar and lacks cellular nutrients or antioxidants that may benefit the conduit. Therefore, the final component of the optimized preparation technique is the use of a balanced, pH-buffered salt solution such as Plasma-Lyte A as the preservation medium, to optimally preserve the graft, particularly the sensitive endothelial monolayer critical to maintenance of graft patency. Components of the OP bundled as a kit are illustrated in Figure 1. A brief demonstration of the OP is available as an mpeg-4 download at https://redcap.vanderbilt.edu/surveys/?s=HWC8NLAXLK, or for streaming at https://www.youtube.com/watch?v=OKogkvaiQ7I. Figure 1 Components of the Optimized Preparation Kit Vein graft preparation represents a unique window of opportunity in which conduit function can be augmented, potentially leading to enhanced vein graft patency. Recognizing an unmet clinical need, an optimized technique has been engineered to prepare HSV graft prior to implantation into the aortocoronary or peripheral circulation. The technique is effective at identifying leaks, maintaining graft orientation and dilating the conduit while requiring only simple modifications from current practice. In vivo studies are currently underway to determine whether there is concomitant improvement in intimal hyperplasia, using a porcine carotid interposition model.

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.

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.

Background: Endothelial dysfunction decreases nitric oxide, which leads to the development of intimal hyperplasia and, eventually, vein graft failure. Failed vein grafts have been found to have calcium deposits, enlarged intima, atherosclerotic plaques, and extracellular matrix deposition. This project examined the effect of calcification on endothelial function by measuring the endothelial relaxation and the expression of nitric oxide synthase (eNOS) in saphenous veins. Methods: Freshly collected HSV (58) samples from CABG patients were equilibrated in a muscle bath and phenylephrine-induced contraction and carbachol-induced relaxation (endothelium-dependent) were measured. Vein sections were stained using Verhoeff-Van Gieson (VVG), Movat, and Von Kossa stains. Endothelial coverage was determined by eNOS, and Lectin staining. Calcification, intimal and medial basal thickness, endothelial coverage, and extracellular matrix deposition data were quantified using light microscopy and digital image analysis. Results: Of the 58 veins analyzed, 17 displayed calcification. The average basal intimal thickness of the samples was 65.44 ± 7.99 μm. Presence of calcification in HSV correlated with higher intimal basal thickness (p=0.0092, n=46, panel A), lower endothelial relaxation (p=0.032, n=43, panel B), loss of eNOS expression (p=0.0397, n=29), loss of endothelial coverage (p=0.0178, n=29), and increased extracellular matrix deposition (p=0.0180, n=22, panel C). Conclusions: Calcified veins displayed lowered endothelial relaxation, higher intimal basal thickness, reduced endothelial coverage, reduced eNOS expression, and increased extracellular matrix deposition. Calcification was associated with factors that contribute to intimal hyperplasia formation and 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 rennin–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 rennin–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 rennin–angiotensin system in the pathogenesis of intimal hyperplasia.

Purpose This investigation examined the trends for gender-based advancement in academic surgery by performing a comparative analysis of the rate of change in the percentage of medical students, surgery residents, and full professors of surgery who are women. Methods All available Women in Medicine Annual Reports were obtained from the American Association of Medical Colleges (AAMC). The gender compositions of medical graduates, surgery residents, and full professors were plotted. Binomial and linear trendlines were calculated to estimate the year when 50% of surgery full professors would be women. Additionally, the percentage distribution of men and women at each professorial rank was determined from 1995 to 2009 using these reports to demonstrate the rate of academic advancement of each gender. Results The slope of the line of increase for women full professors is significantly less than for female medical students and for female general surgery residents (0.36, compared with 0.75 and 0.99, respectively). This predicts that the earliest time that females will account for 50% of full professors in surgery is the year 2096. When comparing women and men in academic ranks, we find that women are much less likely than men to be full professors. Conclusions The percentage of full professors in surgery who are women is increasing at a rate disproportionately slower than the increases in female medical students and surgery residents. The rates of increase in female medical students and surgery residents are similar. The disproportionately slow rate of increase in the number of female full professors suggests that multiple factors may be responsible for this discrepancy.

ObjectiveThis 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).MethodsThe contractile responses (stress, 105 N/m2) 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.ResultsContractile responses varied greatly in HSV harvested for autologous vascular and coronary bypass procedures (0.04198 ± 0.008128 × 105 N/m2 to 0.1192 ± 0.02776 × 105 N/m2). 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).ConclusionsThere 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.Clinical RelevanceHuman great saphenous vein remains the most commonly used conduit for coronary artery bypass grafting and peripheral vascular revascularization. The techniques commonly used for surgical saphenous vein harvest can produce significant injury to the conduit and elicit an exuberant physiologic healing response in the vessel walls, leading to the development of intimal hyperplasia. Intimal hyperplasia is thought to involve smooth muscle proliferation, migration, phenotypic modulation, and extracellular matrix deposition, and ultimately, lead to graft occlusion. This study shows that mechanical stretching injures vascular smooth muscle and reduces contractile responsiveness of the saphenous vein. Thus, by understanding how these injuries can be minimized, mortality and costs associated with vein graft failure can be reduced.

ObjectiveThe present study was aimed at developing a new cell-permeant peptide inhibitor (MK2i) of the kinase that phosphorylates and activates heat-shock protein (HSP)27 (MAPKAP kinase II), and evaluating the ability of this peptide to inhibit HSP27 phosphorylation and intimal thickening.MethodsThe ability of MK2i to reduce HSP27 phosphorylation and cell migration was evaluated in A7R5 cells stimulated with arsenite or lysophosphatidic acid. Stable isotopic labeling using amino acids in cell culture, in combination with liquid chromatography mass spectrometry, was used to characterize the effect of MK2i on global protein expression in fibroblasts. The effect of MK2i on intimal thickening and connective tissue growth factor expression was evaluated in human saphenous vein (HSV) rings maintained with 30% fetal bovine serum for 14 days by light microscopy and immunoblotting.ResultsPretreatment of cells with MK2i (10 μM) prior to arsenite or lysophosphatidic acid stimulation decreased phosphorylation of HSP27 (36% ± 9% and 33% ± 10%, respectively) compared with control (not pretreated) cells. MK2i also inhibited A7R5 migration, and downregulated the transforming growth factor-induced expression of collagen and fibronectin in keloid cells, two major matrix proteins involved in the development of intimal hyperplasia. Treatment of HSV segments with MK2i enhanced relaxation, reduced HSP27 phosphorylation (40% ± 17%), connective tissue growth factor expression (17% ± 5%), and intimal thickness (48.2% ± 10.5%) compared with untreated segments. On the other hand, treatment with a recombinant fusion protein containing a cell-permeant peptide attached to the HSP27 sequence increased intimal thickness of HSV segments by 48% ± 14%.ConclusionOur results suggest that HSP27 may play a role in the development of processes leading to intimal hyperplasia in HSV, and reduction of HSP27 phosphorylation by MK2i may be a potential strategy to inhibit the development of intimal hyperplasia in HSV to prevent the autologous vascular graft failure.Clinical RelevanceIntimal hyperplasia represents the leading cause of prosthetic and autologous vascular graft failure. In spite of the many recent technological advances in vascular interventions, intimal hyperplasia remains an expensive, morbid, and unsolved problem, and this study presents a new strategy in the battle against such a disorder. Cell-permeant peptide-based therapeutics was able to target MAPKAP kinase II and reduce HSP27 phosphorylation, and as a result, prevent the development of intimal hyperplasia. This peptide acts downstream in the signaling cascade compared with other compounds, and therefore may generate less adverse effects. Vein grafts represent an ideal target for cell-permeant peptide therapeutics in that the grafts can be treated ex vivo at the time of surgery. Recent data suggest that the peptides are present intracellularly for up to 7 days in cultured fibroblasts.

BACKGROUND: Human saphenous vein (HSV) remains the most widely utilized conduit for vascular bypass procedures. Outcomes remain limited by vein graft failure and intimal hyperplasia. Endothelial function is considered an important determinant of vein graft failure. We have observed significant variability in endothelial function in freshly isolated HSV samples. We therefore evaluated clinical predictors of endothelial function in HSV. Methods: We obtained freshly isolated HSV samples from the operating room immediately following harvest from patients undergoing coronary artery bypass procedures from Vanderbilt University Hospital and the Nashville VA Hospital. We obtained HSV samples prior to any intraoperative manipulations. HSV viability, smooth muscle function, and endothelial-dependent relaxation (EDR) were measured in a muscle bath. We collected the following clinical and demographic information: age, height, weight, gender, ethnicity, medical comorbidities, laboratory values (creatinine, HbA1c, lipids), ejection fraction, preoperative medication regimen, and method of vein graft harvest. We performed a univariate and multivariate analysis of predictors of endothelial function in HSV. Results: HSV samples were obtained from 149 patients. HSV EDR varied from -11% to 63%. Open harvest was employed for 39 HSV samples, compared with endoscopic harvest in 110 HSV samples. On univariate analysis, only open harvest was a statistically significant predictor of endothelial function (p=0.02): mean HSV EDR was 21.5% in HSV samples harvested open, compared with 15.4% in HSV harvested with an endoscopic technique. HSV EDR was also improved with preoperative aspirin use: mean HSV EDR was 18% in patients with preoperative aspirin use compared with 11.6% in patients who were not administered preoperative aspirin in a multivariate model when controlling for method of vein graft harvest (p=0.05). Conclusions: Endoscopic vein graft harvest is associated with endothelial dysfunction in HSV, while preoperative aspirin use is associated with improved endothelial function. Further work will be necessary to determine whether these factors are associated with development of intimal hyperplasia and vein graft failure.

Background Human saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstruction. Injury during harvest leads to vasospasm and a thrombogenic endoluminal surface. A proteomic transduction approach was developed to prevent vein graft vasospasm and thrombosis. Methods Recombinant HSP20 protein linked to the TAT protein transduction domain was generated in a bacterial expression system (TAT-HSP20). The effect of this protein on the inhibition of smooth muscle contraction was determined using rings of rabbit aorta and HSV in a muscle bath. In addition, the effects of TAT-HSP20 on platelet aggregation were determined in vitro using human citrated whole blood. Results Recombinant TAT-HSP20 inhibited norepinephrine-induced contraction of rabbit aortic and HSV segments. Similarly, TAT-HSP20 induced smooth muscle relaxation in HSV segments precontracted with norepinephrine. In human-citrated whole blood, platelet aggregation was significantly inhibited by TAT-HSP20 in a dose-dependent manner. Conclusions The results of this study demonstrate that recombinant TAT-HSP20 inhibits vascular smooth muscle contraction and platelet aggregation. This suggests that HSP20 may be an ideal effector molecule to target as a proteomic approach to enhance early vein graft patency rates by preventing acute vasospasm and thrombosis.

BackgroundHuman saphenous vein (HSV) is the autologous conduit of choice for peripheral vascular reconstructions. However, vasospasm can lead to early graft failure. The leading cause of delayed graft failure is intimal hyperplasia.ObjectiveTo develop a proteomic approach to prevent vein-graft spasm and intimal hyperplasia.MethodsBiomimetic peptide analogs of the small heat shock–related protein HSP20, containing a protein transduction domain (PTD), a phosphorylated serine, and a sequence of HSP20 surrounding the phosphorylation site (PTD-pHSP20), or a scrambled sequence of the same amino acids surrounding the phosphorylation site (PTD-scHSP20) were synthesized. The peptides were used in muscle bath and organ culture experiments with human saphenous vein (HSV) segments. Cultured smooth muscle cell lines were used to determine the effect of the peptides on proliferation and migration.ResultsIn HSV rings precontracted with norepinephrine, PTD-pHSP20 but not PTD-scHSP20 led to relaxation. There was no significant difference in smooth muscle cell proliferation in cells treated with PTD-pHSP20 compared with PTD-scHSP20. Treatment with PTD-pHSP20 significantly inhibited cellular migration compared with PTD-scHSP20. Control, untreated, and PTD-scHSP20–treated saphenous veins had significant increases in intimal thickness after culture. This intimal thickening was completely inhibited by treatment with PTD-pHSP20.ConclusionsProtein transduction of biologically active motifs of HSP20 can affect pathologic and physiologic responses of HSV and represents a novel proteomic-based therapeutic approach.AbstractClinical relevanceWe have been a part of the genomics era and are now viewing the emergence of “proteomics.” The genome is linear and relatively easy to examine; however the proteome is much more complex and dynamic. In essence, the purpose of gene therapy is to manipulate the genome to produce a particular protein. This manuscript describes a new proteomic approach in which the biologically active part of a protein is directly introduced into vascular cells. Peptides were synthesized which contained a total of 24 amino acids, 11 of which represent a protein transduction domain or “carrier” while the other 13 are the biologically active “cargo.” These synthetic peptides prevent spasm (contraction) and intimal hyperplasia in segments of human saphenous vein treated ex vivo. Preclinical development is currently underway to develop these molecules as a proteomic-based vein harvest solution to enhance vein-graft patency.

Purpose Sildenafil is an oral phosphodiesterase type 5 inhibitor that is a vasodilator used in the treatment of erectile dysfunction. Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of the heat shock related protein 20 (HSP20). The purpose of this study was to determine if sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of HSP20. Materials and methods Peptides containing an 11 amino acid enhanced protein transduction domain (PTD) and the functional 13 amino acid sequence of HSP20 with a phosphoserine (PTD-pHSP20) were synthesized using F-MOC technology. Rings of porcine coronary artery were suspended in a muscle bath and sub-maximally contracted with serotonin. Increasing concentrations of sodium nitroprusside (SNP; 0.01–10 μ m ), sildenafil (0.01–100 μ m ), or PTD-pHSP20 (0.1–1.0 m m ) were added to the baths and the percent relaxation was recorded. To determine if sildenafil-induced vasorelaxation was associated with increases in the phosphorylation of HSP20, rings of porcine coronary artery were untreated (control) or treated with SNP (10 μ m ) or sildenafil (100 μ m ) for 2, 5, and 10 min and then snap frozen. Extracted proteins were then separated using two-dimensional SDS-PAGE, transferred to a membrane, and probed for HSP20. Results Sildenafil induced vasorelaxation of pre-contracted coronary artery in a dose-dependent manner. Sildenafil-induced vasorelaxation was associated with an increase in the phosphorylation of HSP20. Transduction of peptide analogues of pHSP20 led to a dose-dependent relaxation of pre-contracted porcine coronary artery. Conclusions These findings suggest that sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of HSP20 and that transduction of phosphopeptide analogues of HSP20 is sufficient for relaxation of vascular smooth muscle.

Cyclic nucleotide-dependent relaxation of vascular smooth muscle is associated with increases in the phosphorylation of the small heat shock—related protein, HSP20. To determine whether phosphorylated HSP20 directly mediates relaxation, we used gene transfection and protein transduction of HSP20 analogues. Rat mesangial cells were transfected with constructs containing wild-type HSP20-enhanced green fluorescent protein (EGFP), phosphorylation site mutated HSP20 (S16A-HSP20-EGFP), or EGFP alone. Contractile properties were determined on a silicone polymer substrata. In the presence of serum, EGFP-vector transfected control cells and S16A-HSP20 transfected cells formed wrinkles on the polymer (contracted). Activation of cyclic nucleotide signaling pathways in the EGFP-vector transfected control cells led to a time-dependent decrease in the wrinkles (relaxation). The S16A-HSP20 transfected cells were refractory to cyclic nucleotide-dependent relaxation. Cells overexpressing the wild-type HSP20 did not form wrinkles on the polymer in response to serum (refractory to contraction). Treatment of precontracted strips of intact bovine carotid artery smooth muscle with synthetic peptides containing HIV-trans-activating transcriptional activator and a phosphopeptide motif of HSP20 led to dose-dependent relaxation. These data provide evidence that phosphorylated HSP20 has a direct role in smooth muscle relaxation and that small phosphopeptide motifs of HSP20 can mimic the effects of the entire molecule. (J Vasc Surg 2003;37:874-81.)

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.

Surgical skin markers are used off-label to mark human saphenous veins (HSVs) to maintain orientation before implantation as aortocoronary or peripheral arterial bypass grafts. These surgical skin markers impair functional responses of the HSV tissue.To investigate the effect of brilliant blue dye 1 (brilliant blue FCF [for food coloring]; hereinafter, FCF) as a nontoxic alternative marking dye and to determine whether FCF has pharmacological properties.Segments of HSVs were collected in university hospitals from patients undergoing coronary artery bypass grafting procedures immediately after harvest (unmanipulated) or after typical intraoperative surgical graft preparation (after manipulation). Rat inferior venae cavae were used to determine the pharmacological properties and cellular targets of FCF. Endothelial and smooth muscle functional responses were determined in a muscle bath, and intimal thickening in HSVs was determined after 14 days in organ culture.Contractile responses were measured in force and converted to stress. Smooth muscle function was expressed as maximal responses to potassium chloride depolarization contractions. Endothelial function was defined as the percentage of relaxation of maximal agonist-induced contraction. Neointimal thickness was measured by histomorphometric analysis.Human saphenous veins stored in the presence of FCF had no loss of endothelial or smooth muscle function. Unmanipulated HSVs preserved in the presence of FCF demonstrated a significant increase in endothelial-dependent relaxation (mean [SEM], 25.2% [6.4%] vs 30.2% [6.7%]; P = .02). Application of FCF to functionally nonviable tissue significantly enhanced the smooth muscle responses (mean [SEM], 0.018 [0.004] × 10⁵ N/m² vs 0.057 [0.016] × 10⁵ N/m²; P = .05). Treatment with FCF reduced intimal thickness in organ culture (mean [SEM], -17.5% [2.1%] for unmanipulated HSVs vs -27.9% [3.7%] for HSVs after manipulation; P .001). In rat inferior venae cavae, FCF inhibited the contraction induced by the P2X7 receptor agonist 2'(3')-O-(4-benzoyl)benzoyl-adenosine-5'-triphosphate (mean [SEM], 14.8% [2.2%] vs 6.5% [1.8%]; P = .02) to an extent similar to the P2X7 receptor antagonist oxidized adenosine triphosphate (mean [SEM], 5.0% [0.9%]; P .02 vs control) or the pannexin hemichannel inhibitor probenecid (mean [SEM], 7.3% [1.6%] and 4.7% [0.9%] for 0.5mM and 2mM, respectively; P .05).Treatment with FCF did not impair endothelial or smooth muscle function in HSVs. Brilliant blue FCF enhanced endothelial-dependent relaxation, restored smooth muscle function, and prevented intimal hyperplasia in HSVs in organ culture. These pharmacological properties of FCF may be due to P2X7 receptor or pannexin channel inhibition. Brilliant blue FCF is an alternative, nontoxic marking dye that may improve HSV conduit function and decrease intimal hyperplasia.

Long-term patency of human saphenous veins (HSVs) used as autologous conduits for coronary artery bypass grafting (CABG) procedures remains limited because of vein graft failure (VGF). Vein graft failure has been reported to be as high as 45% at 12 to 18 months after surgery and leads to additional surgery, myocardial infarction, recurrent angina, and death. Preparation of HSVs before implantation leads to conduit injury, which may promote VGF.To investigate whether pressure distension during vein graft preparation leads to endothelial injury and intimal thickening and whether limiting intraluminal pressure during pressure distension by using a pressure release valve (PRV) preserves endothelial function and prevents neointima thickening.Segments of HSVs were collected in a university hospital from 13 patients undergoing CABG procedures immediately after harvest (unmanipulated [UM]), after pressure distension (after distension [AD]), and after typical intraoperative surgical graft preparation (after manipulation [AM]). Porcine saphenous veins (PSVs) from 7 healthy research animals were subjected to manual pressure distension with or without an in-line PRV that prevents pressures of 140 mm Hg or greater. Endothelial function of the HSVs and PSVs was determined in a muscle bath, endothelial integrity was assessed, and intimal thickening in PSVs was evaluated after 14 days in organ culture.Endothelial function was measured in force, converted to stress, and defined as the percentage relaxation of maximal phenylephrine-induced contraction. Endothelial integrity was assessed by immunohistologic examination. Neointimal thickness was measured by histomorphometric analysis.Pressure distension of HSVs led to decreased mean (SEM) endothelial-dependent relaxation (5.3% [2.3%] for AD patients vs 13.7% [2.5%] for UM patients; P .05) and denudation. In the AM group, the function of the conduits was further decreased (-3.2% [3.2%]; P .05). Distension of the PSVs led to reduced endothelial-dependent relaxation (7.6% [4.4%] vs 61.9% [10.2%] in the control group; P .05), denudation, and enhanced intimal thickening (15.0 [1.4] µm vs 2.2 [0.8] µm in the control group; P .05). Distension with the PRV preserved endothelial-dependent relaxation (50.3% [9.6%]; P = .32 vs control), prevented denudation, and reduced intimal thickening (3.4 [0.8] µm; P = .56 vs controls) in PSVs.Use of a PRV during graft preparation limits intraluminal pressure generated by manual distension, preserves endothelial integrity, and reduces intimal hyperplasia. Integration of this simple device may contribute to improved long-term vein graft patency.

Severe acute asthma, defined as asthma that is refractory to current anti-inflammatory agents and bronchodilators, accounts for an estimated two-million emergency department visits annually. β2-adrenergic receptor (β2AR) agonists (β-agonists) are widely used for the treatment of bronchospasm associated with acute asthma attacks, however, some patients develop refractory responses to long term usage of β –agonists. It also causes adverse effects in patients with a genetic polymorphism (Arg/Arg genotype, about a sixth of the US population) in the adrenergic receptor creating a need for additional therapeutics for managing asthma. We have shown earlier that β-agonist induced airway smooth muscle (ASM) relaxation is associated with increases in the phosphorylation of HSP20. In this study we evaluate the effect of a transducible phosphopeptide mimetic of HSP20 protein (P20 peptide) on the relaxation of human ASM. Freshly isolated human ASM was pretreated with either a β-agonist, isoproterenol (ISO), or the P-2...

Outcomes from vein graft bypass are limited by graft failure, leading causes of which include intimal hyperplasia and vasospasm. intimal hyperplasia remains the most common cause of graft failure, but no therapeutic modalities have been shown to prevent intimal hyperplasia in humans. The small heat shock proteins are a class of naturally occurring proteins in vascular smooth muscle. These proteins have an integral role in maintenance of vascular tone and in cellular defense against various stressors. Transduction domains have enabled intracellular therapeutic delivery of peptide analogs of heat shock proteins, as well as peptide inhibitors of the kinases that phosphorylate these proteins. These cell-permeant peptides have been shown to prevent vasospasm and intimal hyperplasia in vitro. Since vascular bypass using vein grafts is analogous to autologous organ transplantation, ex vivo treatment of the vein graft with cell-permeant peptide inhibitors of vasospasm and intimal hyperplasia prior to implantation provides a unique opportunity for targeted treatment of the graft to improve patency.

Background Vasospasm occurs in conduits used for vascular reconstructions. The small heat shock proteins, HSP20 and HSP27, coordinately regulate vascular smooth muscle tone. Phosphorylated HSP20 is associated with vasorelaxation, and phosphorylated HSP27 inhibits the phosphorylation of HSP20 and relaxation. We hypothesized that the relationship between the phosphorylated states of these two proteins might dictate the tone of a vessel and may contribute to vasospasm. Study design Sodium nitroprusside relaxation of vascular smooth muscle was recorded using pig coronary artery and human saphenous vein. Segments were frozen and homogenized, and extracted proteins were separated by one- and two-dimensional gel electrophoresis, transferred to Immobilon (Millipore), and probed with anti-cGMP-dependent protein kinase (anti-PKG), -HSP20, -HSP27, and -phosphoHSP27 antibodies. Band intensity was estimated using densitometry. Results Pig coronary artery completely relaxed (100%) with SNP (10 −7 M), but human saphenous vein only partially relaxed (20%). The levels of cGMP-dependent protein kinase and HSP20 were similar in the two tissue types. Human saphenous vein had significantly higher levels of HSP27 versus pig coronary artery (30.14 ± 0.8 versus 6.62 ± 0.2 pixels/mg; p ≤ 0.001) and phosphoHSP27 (8.29 ± 3.43 versus 0.012 ± 0.008 pixels/mg; p ≤ 0.001). Conclusions Human saphenous vein contained significantly higher levels of HSP27 and pHSP27. Increased levels of phosphorylated HSP27 might contribute to vasospasm in human saphenous vein.

Introduction: HSP27 and HSP20, small heat shock proteins, have been implicated in the regulation of vascular smooth muscle (VSM) tone. Stress induced hypertension leads to increased expression of HSP27 in VSM. Transduction of VSM with HSP20 leads to vasorelaxation. Phosphorylated HSP27 (pHSP27) inhibits the activation of HSP20. We hypothesize that expression of HSP27 is greater in VSM refractory to cyclic nucleotide vasorelaxation. Methods: Sodium nitroprusside (SNP 10-7M) induced relaxation of VSM was measured using segments of pig coronary artery (PCA) and human saphenous vein (HSV) in a muscle bath. The levels of PKG, HSP20, phosphorylated HPS20 (pHSP20), HSP27 and pHSP27 in tissue extracts were determined by one- and two-dimensional gel electrophoresis and western blotting. Band intensity was estimated by densitometry. Results: PCA completely relaxed with SNP (100%), however, HSV did not (60%). Both tissue types had similar levels of expression of PKG and HSP20. The levels of HSP27 were 5 fold higher in the HSV (6.62 + 0.15 vs. 30.14 + 0.80) and pHSP27 was undetectable in PCA (0 vs.2.25 + 0.1). The levels of pHSP20 were undetectable in HSV. Conclusions: HSV segments refractory to relaxation with SNP had similar levels of expression of PKG and HSP20 as PCA segments demonstrating that the mediators of cyclic nucleotide vasorelaxation were present in both tissues. Increased levels of pHSP27 inhibit the phosphorylation of HSP20, and therefore, vasorelaxation of the HSV segments. Intracellular pHSP20 may be necessary and sufficient for VSM relaxation.