Nicole L. Lohr

Chronic Hyperglycemia Attenuates Coronary Collateral Development and Impairs Proliferative Properties of Myocardial Interstitial Fluid by Production of Angiostatin

Background—Development of coronary collateral vessels is impaired in patients with diabetes mellitus. We tested the hypothesis that hyperglycemia alone attenuates collateral development and abolishes proliferative properties of myocardial interstitial fluid (MIF) by enhancing expression of matrix metalloproteinases (MMP) and angiostatin. Methods and Results—Chronically instrumented dogs were randomly assigned to receive an infusion of normal saline (control; n=9) or 70% dextrose in water to increase blood glucose to 350 to 400 mg/dL for 8 h/d (hyperglycemia; n=7) in the presence or absence (sham; n=9) of brief (2 minutes), repetitive coronary artery occlusions (1/h; 8/d for 21 days). Collateral perfusion increased to 41±11% and 49±6% of normal zone flow in control dogs on days 14 and 21 (P <0.05) but remained unchanged over 21 days in hyperglycemic and sham dogs (12±3% and 13±3%, respectively). A progressive reduction of the postocclusive peak reactive hyperemic response was also observed in control dogs (16±1 to 10±1 Hz · 102 on days 1 and 21, respectively) but not in hyperglycemic (17±2 to 20±2) or sham (17±2 to 16±1) dogs. Endothelial cell tube formation was produced by MIF obtained from control dogs but not hyperglycemic or sham dogs. Coincubation of MIF from hyperglycemic dogs with an angiostatin antibody restored endothelial cell tube formation. MMP-9 activity and expression of angiostatin were increased in dogs receiving exogenous glucose compared with controls Conclusions—Chronic hyperglycemia abolishes development of coronary collateral vessels by increasing MMP-9 activity and angiostatin expression in dogs.

Enhancement of nitric oxide release from nitrosyl hemoglobin and nitrosyl myoglobin by red/near infrared radiation: Potential role in cardioprotection

Nitric oxide is an important messenger in numerous biological processes, such as angiogenesis, hypoxic vasodilation, and cardioprotection. Although nitric oxide synthases (NOS) produce the bulk of NO, there is increasing interest in NOS independent generation of NO in vivo, particularly during hypoxia or anoxia, where low oxygen tensions limit NOS activity. Interventions that can increase NO bioavailability have significant therapeutic potential. The use of far red and near infrared light (R/NIR) can reduce infarct size, protect neurons from methanol toxicity, and stimulate angiogenesis. How R/NIR modulates these processes in vivo and in vitro is unknown, but it has been suggested that increases in NO levels are involved. In this study we examined if R/NIR light could facilitate the release of NO from nitrosyl heme proteins. In addition, we examined if R/NIR light could enhance the protective effects of nitrite on ischemia and reperfusion injury in the rabbit heart. We show both in purified systems and in myocardium that R/NIR light can decay nitrosyl hemes and release NO, and that this released NO may enhance the cardioprotective effects of nitrite. Thus, the photodissociation to NO and its synergistic effect with sodium nitrite may represent a noninvasive and site specific means for increasing NO bioavailability.

Isoflurane differentially modulates mitochondrial reactive oxygen species production via forward versus reverse electron transport flow: Implications for preconditioning

Background: Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, the authors investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, the authors investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles. Methods: Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2 h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined. Results: Isoflurane reduced myocardial infarct size (40 ± 9% = mean ± SD) compared with control experiments (60 ± 4%). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60 ± 9%). Isoflurane enhanced ROS generation in submitochondrial particles with nicotinamide adenine dinucleotide (reduced form), but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity. Conclusions: The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.

Transient repetitive exposure to low level light therapy enhances collateral blood vessel growth in the ischemic hindlimb of the tight skin mouse.

The tight skin mouse (Tsk−/+) is a model of scleroderma characterized by impaired vasoreactivity, increased oxidative stress, attenuated angiogenic response to VEGF and production of the angiogenesis inhibitor angiostatin. Low‐level light therapy (LLLT) stimulates angiogenesis in myocardial infarction and chemotherapy‐induced mucositis. We hypothesize that repetitive LLLT restores vessel growth in the ischemic hindlimb of Tsk−/+ mice by attenuating angiostatin and enhancing angiomotin effects in vivo. C57Bl/6J and Tsk−/+ mice underwent ligation of the femoral artery. Relative blood flow to the foot was measured using a laser Doppler imager. Tsk−/+ mice received LLLT (670 nm, 50 mW cm−2, 30 J cm−2) for 10 min per day for 14 days. Vascular density was determined using lycopersicom lectin staining. Immunofluorescent labeling, Western blot analysis and immunoprecipitation were used to determine angiostatin and angiomotin expression. Recovery of blood flow to the ischemic limb was reduced in Tsk−/+ compared with C57Bl/6 mice 2 weeks after surgery. LLLT treatment of Tsk−/+ mice restored blood flow to levels observed in C57Bl/6 mice. Vascular density was decreased, angiostatin expression was enhanced and angiomotin depressed in the ischemic hindlimb of Tsk−/+ mice. LLLT treatment reversed these abnormalities. LLLT stimulates angiogenesis by increasing angiomotin and decreasing angiostatin expression in the ischemic hindlimb of Tsk−/+ mice.

Far Red/Near Infrared Light Treatment Promotes Femoral Artery Collateralization in the Ischemic Hindlimb

Nitric oxide (NO) is a crucial mediator of hindlimb collateralization and angiogenesis. Within tissues there are nitrosyl-heme proteins which have the potential to generate NO under conditions of hypoxia or low pH. Low level irradiation of blood and muscle with light in the far red/near infrared spectrum (670 nm, R/NIR) facilitates NO release. Therefore, we assessed the impact of red light exposure on the stimulation of femoral artery collateralization. Rabbits and mice underwent unilateral resection of the femoral artery and chronic R/NIR treatment. The direct NO scavenger carboxy-PTIO and the nitric oxide synthase (NOS) inhibitor L-NAME were also administered in the presence of R/NIR. DAF fluorescence assessed R/NIR changes in NO levels within endothelial cells. In vitro measures of R/NIR induced angiogenesis were assessed by endothelial cell proliferation and migration. R/NIR significantly increased collateral vessel number which could not be attenuated with L-NAME. R/NIR induced collateralization was abolished with c-PTIO. In vitro, NO production increased in endothelial cells with R/NIR exposure, and this finding was independent of NOS inhibition. Similarly R/NIR induced proliferation and tube formation in a NO dependent manner. Finally, nitrite supplementation accelerated R/NIR collateralization in wild type C57Bl/6 mice. In an eNOS deficient transgenic mouse model, R/NIR restores collateral development. In conclusion, R/NIR increases NO levels independent of NOS activity, and leads to the observed enhancement of hindlimb collateralization.

An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice

Interferon regulatory factor 5 (IRF5) has been called a “master switch” for its ability to determine whether cells mount proinflammatory or anti-inflammatory responses. Accordingly, IRF5 should be an attractive target for therapeutic drug development. Here we report on the development of a novel decoy peptide inhibitor of IRF5 that decreases myocardial inflammation and improves vascular endothelial cell (EC) function in tight-skin (Tsk/+) mice. Biolayer interferometry studies showed the Kd of IRF5D for recombinant IRF5 to be 3.72 ± 0.74x10-6M. Increasing concentrations of IRF5D (0–100 μg/mL, 24h) had no significant effect on EC proliferation or apoptosis. Treatment of Tsk/+ mice with IRF5D (1mg/kg/d subcutaneously, 21d) reduced IRF5 and ICAM-1 expression and monocyte/macrophage and neutrophil counts in Tsk/+ hearts compared to expression in hearts from PBS-treated Tsk/+ mice (p<0.05). EC-dependent vasodilatation of facialis arteries isolated from PBS-treated Tsk/+ mice was reduced (~15%). IRF5D treatments (1mg/kg/d, 21d) improved vasodilatation in arteries isolated from Tsk/+ mice nearly 3-fold (~45%, p<0.05), representing nearly 83% of the vasodilatation in arteries isolated from C57Bl/6J mice (~55%). IRF5D (50μg/mL, 24h) reduced nuclear translocation of IRF5 in myocytes cultured on both Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix (p<0.05). These data suggest that IRF5 plays a causal role in inflammation, fibrosis and impaired vascular EC function in Tsk/+ mice and that treatment with IRF5D effectively counters IRF5-dependent mechanisms of inflammation and fibrosis in the myocardium in these mice.

Man Overboard!Rescuing Myocardium with Membrane Rafts

Volatile anesthetics produce important cardioprotective effects by stimulating a series of intracellular signaling events that ultimately render myocardium resistant to infarction. Anesthetics are known to protect the heart in a temporal manner. An initial early window of myocardial protection lasts hours after exposure to isoflurane or other volatile agents, and myocardial protection reappears again 24 to 48 h later. The mechanisms of early and delayed anesthetic preconditioning differ. Anesthetics activate various intracellular kinases which phosphorylate and subsequently modify the activity of downstream proteins (e.g., endothelial nitric oxide synthase [eNOS] and adenosine triphosphate-regulated potassium channels) that are important in mediating cardioprotection. During the early preconditioning phase, modification of preexisting proteins leads to protection, whereas after 24 h, cardioprotection relies on the synthesis of new proteins. The complexity of these signal transduction events requires both functional and spatial organization, and coordination of the activity of a large number of intracellular proteins. In this issue of the Journal, Tsutsumi et al1 demonstrate that isoflurane produces delayed protection against myocardial infarction by modulating a key protein, caveolin-3, found in membrane (lipid) rafts (fig. 1). An extension of the classical fluid lipid bilayer model of the plasma membrane, lipid/membrane rafts are small (10–200 nm) microdomains enriched in sterols, sphingolipids and cholesterol “floating” in a sea of phospholipids.2 These lipid domains form docking platforms that control the location of intracellular signal transduction events. Rafts are located in the plasma membrane, and are also found in the endoplasmic reticulum and mitochondria. Membrane rafts function to regulate cellular processes by concentrating proteins to highly specific intracellular locations. The formation of lipid rafts is highly dynamic and this property allows for temporal regulation of protein signaling and trafficking. A subclass of membrane rafts are the caveolae, which are flask-like invaginations of the cellular membrane (60–80 nm), distinguished by the presence of scaffolding proteins caveolin-1, -2, and -3.3 Caveolins-1 and -2 are highly expressed in adipocytes, endothelial cells, and fibroblasts, whereas, caveolin-3 is expressed predominantly in skeletal, cardiac, and smooth muscle cells. Caveolae are disrupted in caveolin-1 and caveolin-3 knock out mice, but are preserved in caveolin-2 mutants.4 Caveolins bind proteins through a specific domain that enables conformational changes to occur and this action regulates the activity of signal transduction molecules. Caveolins are required for caveolae formation and their expression indirectly regulates the number of caveolae available for functional signal transduction. Caveolins can alter the fluidity of membrane rafts through the binding of cholesterol which in turn alters membrane composition and signaling effects.4 Tsutsumi et al5 have previously shown that cardiac-specific over-expression of caveolin-3 decreases myocardial infarction and mimics ischemic preconditioning. The current results extend these previous findings and demonstrate that isoflurane produces delayed preconditioning by up-regulating caveolin-3 and by increasing the co-localization of caveolin-3 with glucose-transporter (GLUT)-4 in membrane rafts (caveolar fraction). GLUT-4 is the major transporter responsible for glucose uptake into cells. During ischemia, cardiac myocyte metabolism is altered to favor anaerobic glycolysis and increased GLUT-4 translocation from intracellular compartments to the plasma membrane facilitates substrate availability. GLUT-4 has previously been implicated in the cardioprotective effect of both early and delayed forms of ischemic preconditioning, a phenomenon in which brief periods of myocardial ischemia up to 2 (early) or 24–48 h (delayed) before a prolonged period of coronary artery occlusion and reperfusion decreases the extent of subsequent infarction. Myocardial ischemia appears to increase GLUT-4 expression and translocation. GLUT-4 protein is up-regulated after ischemic preconditioning along with increased expression of caveolin-3, phosphorylated eNOS, and phosphorylated Akt. Preconditioning stimuli not only increase the expression of these cardioprotective proteins, but also stimulate translocation of GLUT-4 to the caveolar-rich membrane fraction, thereby, sustaining activation of signaling molecules.6 Interestingly eNOS, which is known to play an important role during both early and delayed phases of anesthetic preconditioning, is reciprocally regulated by interactions with caveolins-1 and -3, and GLUT-4. The activity of eNOS is decreased when this enzyme is associated with caveolin-1. Conversely, disassociation of caveolin-1/eNOS interaction, and increased translocation of GLUT-4 and its enhanced association with caveolin-3 activates eNOS.7 Although caveolin-1 is an important mediator of the early phase of isoflurane preconditioning, there appears to be almost no role for this protein in delayed protection. These findings indicate a differential role of caveolins during anesthetic cardioprotection that may be both temporal (early vs. delayed) and cell lineage (endothelial cell vs. cardiomyocyte) specific. Caveolin-1, while negatively regulating eNOS under basal conditions, also facilitates eNOS signaling during stimulation by compartmentalizing proteins in the appropriate intracellular locations. This condition is referred to as “caveolar paradox” and illustrates the complex nature of protein-protein interactions that ultimately impacts cell survival after ischemia and reperfusion. Other binding proteins, such as heat shock protein-90, have also been implicated in the regulation of eNOS by caveolins. For example, caveolin-1, eNOS, and heat shock protein 90 can be co-immunoprecipitated from endothelial cells, and the presence of heat shock protein 90 decreases the inhibitory effect of caveolin-1 on eNOS activity.8 Tsutsumi et al1 did not investigate eNOS regulation by GLUT-4/caveolin-3 interactions during delayed anesthetic preconditioning; however, it is interesting to speculate that interactions between these molecules and other binding partners, such as heat shock protein 90, may be spatially regulated in caveolae by lipophilic volatile anesthetics. GLUT-4 is the major insulin-responsive glucose transporter. Insulin-induced translocation of GLUT-4 to the membrane from intracellular vesicles stimulates glucose uptake in muscle and adipose tissues. However, the exact mechanism whereby translocation of GLUT-4 to the plasma membrane induces cardioprotection is unknown. The current findings that GLUT-4 translocation is enhanced by delayed preconditioning with isoflurane in caveolin-1 but not -3 knockout mice also implicates caveolins as potential targets of disease processes, such as diabetes, that modulate the efficacy of preconditioning. For example, diabetes and acute hyperglycemia attenuate reduction of myocardial infarct size elicited by diverse preconditioning stimuli and this occurs through impaired eNOS regulation by heat shock protein 90.9 The caveolins are also modulated by diabetes. Caveolin-3 content is reduced in lipid rafts from diabetic myocardium7 and disruption of caveolae in adipocytes renders these cells insulin resistant.10 Caveolin-3 knock-out mice exhibit insulin resistance in vivo, and acute hyperglycemia alone disturbs lipid raft stability by interfering with cholesterol synthesis.11 Thus, defects in membrane raft composition and caveolin expression induced by disease states could underlie the lack of cardioprotection observed in some clinical studies using volatile anesthetics, although, this hypothesis remains to be specifically tested. The work by Tsutsumi and colleagues1 highlighted in this issue of the Journal emphasizes that anesthetic cardioprotection requires functional lipid domains, such as caveolae, and their associated proteins the caveolins. Membrane rafts regulate the intracellular location of signaling molecules activated by volatile anesthetics and these microdomains may be the interface through which anesthetics directly mediate protection. Moreover, lipid rafts may represent a new target for the rescue of ischemic myocardium and provide a new therapeutic approach in the treatment of patients with cardiovascular disease.

Red/near infrared light stimulates release of an endothelium dependent vasodilator and rescues vascular dysfunction in a diabetes model

Abstract Peripheral artery disease (PAD) is a morbid condition whereby ischemic peripheral muscle causes pain and tissue breakdown. Interestingly, PAD risk factors, e.g. diabetes mellitus, cause endothelial dysfunction secondary to decreased nitric oxide (NO) levels, which could explain treatment failures. Previously, we demonstrated 670 nm light (R/NIR) increased NO from nitrosyl‐heme stores, therefore we hypothesized R/NIR can stimulate vasodilation in healthy and diabetic blood vessels. Vasodilation was tested by ex vivo pressure myography in wild type C57Bl/6, endothelial nitric oxide synthase (eNOS) knockout, and db/db mice (10 mW/cm2 for 5 min with 10 min dark period). NOS inhibition with N‐Nitroarginine methyl ester (L‐NAME) or the NO scavenger Carboxy‐PTIO (c‐PTIO) tested the specificity of NO production. 4,5‐Diaminofluorescein diacetate (DAF‐2) measured NO in human dermal microvascular endothelial cells (HMVEC‐d). R/NIR significantly increased vasodilation in wild type and NOS inhibited groups, however R/NIR dilation was totally abolished with c‐PTIO and blood vessel denudation. Interestingly, the bath solution from intact R/NIR stimulated vessels could dilate light naïve vessels in a NO dependent manner. Characterization of the bath identified a NO generating substance suggestive of S‐nitrosothiols or non heme iron nitrosyl complexes. Consistent with the finding of an endothelial source of NO, intracellular NO increased with R/NIR in HMVEC‐d treated with and without L‐NAME (1 mM), yet c‐PTIO (100 &mgr;m) reduced NO production. R/NIR significantly dilated db/db blood vessels. In conclusion, R/NIR stimulates vasodilation by release of NO bound substances from the endothelium. In a diabetes model of endothelial dysfunction, R/NIR restores vasodilation, which lends the potential for new treatments for diabetic vascular disease. Graphical abstract No caption available. Highlights670 nm light dilates blood vessels in an endothelial dependent mechanism.Vasodilation requires nitric oxide but is independent of nitric oxide synthase.Light acts on the endothelium to release a vasodilator substance.S‐nitrosothiol or dinitrosyl iron complexes are candidates for this vasodilator.Light is an effective vasodilator in endothelial dysfunction, e.g. diabetes mellitus.

Collateral Development The Quest Continues

The promise of stimulating collateral blood vessel growth in the heart, brain, or peripheral circulation remains a holy grail in the field of vascular biology. For decades, clinicians and researchers have observed some individuals remain asymptomatic, despite highly obstructive atherosclerotic disease because they possess well-developed innate collateral vessels.1–4 To date, we stand on a mountain of evidence supporting their existence, their potential to be modified by various stimuli (eg, shear stress and ischemia), and their clinical benefits.5–7 Yet, we incompletely understand how much of collateral development depends on the genetic composition of a patient, and how much it is related to the environment in which these vessels are exposed. Article, see p 660 A functional collateral circulation is the end result of 2 different processes: collaterogenesis and arteriogenesis. During embryogenesis, arterial–arterial connections at the level of the microcirculation form within the heart, brain, and skeletal muscle. In the brain, this mechanism occurs through the direct sprouting and proliferation of endothelial cells to form tubes that directly attach to a neighboring arteriole.8 Vascular endothelial growth factor (VEGF) seems to mediate this endothelial migration and proliferation through its receptor, flk-1, and Notch signal pathway.9 VEGF levels were directly related to the extent of native collateral growth in the brain and skeletal muscles when measured in different mouse strains.10,11 Interestingly, an expression quantitative trait locus (QTL) on chromosome 17 near vegfa was identified as a possible cause for the strain-dependent variation in VEGF expression.11 This …

Ascorbate attenuates red light mediated vasodilation: Potential role of S-nitrosothiols

There is significant therapeutic advantage of nitric oxide synthase (NOS) independent nitric oxide (NO) production in maladies where endothelium, and thereby NOS, is dysfunctional. Electromagnetic radiation in the red and near infrared region has been shown to stimulate NOS-independent but NO-dependent vasodilation, and thereby has significant therapeutic potential. We have recently shown that red light induces acute vasodilatation in the pre-constricted murine facial artery via the release of an endothelium derived substance. In this study we have investigated the mechanism of vasodilatation and conclude that 670 nm light stimulates vasodilator release from an endothelial store, and that this vasodilator has the characteristics of an S-nitrosothiol (RSNO). This study shows that 670 nm irradiation can be used as a targeted and non-invasive means to release biologically relevant amounts of vasodilator from endothelial stores. This raises the possibility that these stores can be pharmacologically built-up in pathological situations to improve the efficacy of red light treatment. This strategy may overcome eNOS dysfunction in peripheral vascular pathologies for the improvement of vascular health.