cyclic-gmp and Ischemia

Although an abundant amount of research has been devoted to the study of angiogenesis, its precise mechanisms are incompletely understood. Numerous clinical trials focused on therapeutic angiogenesis for the treatment of tissue ischaemia have not been as successful as those of preclinical studies. Thus, additional studies are needed to better understand critical molecular mechanisms regulating ischaemic neovascularization to identify novel therapeutic agents. Nitric oxide (NO) plays a central role in ischaemic neovascularization through the generation of cyclic guanosine monophosphate (cGMP) and the activation of several other signalling responses. Accumulated evidence suggests that endothelial protein kinase A/endothelial NO synthase (PKA/eNOS) signalling may play an important role in ischaemic disorders by promoting neovascularization. This review highlights recent advances in the role of the PKA/eNOS and NO-cGMP-kinase cascade pathway in ischaemic neovascularization. We also discuss molecular relationships of PKA/eNOS with other angiogenic pathways and explore the possibility of activation of the NO/nitrite endocrine system as potential therapeutic targets for ischaemic angiogenesis.

Topics: Animals; Atherosclerosis; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Humans; Ischemia; Neovascularization, Physiologic; Nitric Oxide; Nitric Oxide Synthase Type III; Reactive Oxygen Species; Signal Transduction

Nitric oxide synthase (NOS), the enzyme that catalyzes the formation of nitric oxide from L-arginine, exists in three major isoforms, neuronal, endothelial, and immunologic. Neuronal and endothelial isoforms are constitutively expressed, and require calcium for activation. Both of these isoforms can be induced (i.e., new protein synthesis occurs) under appropriate conditions. The immunologic isoform is not constitutively expressed, and requires induction usually by immunologic activation; calcium is not necessary for its activation. Neuronal and immunologic NOS have been detected in the retina. Neuronal NOS may be responsible for producing nitric oxide in photoreceptors and bipolar cells. Nitric oxide stimulates guanylate cyclase of photoreceptor rod cells and increases calcium channel currents. In the retina of cats, NOS inhibition impairs phototransduction as assessed by the electroretinogram. Inducible nitric oxide synthase, found in Müller cells and in retinal pigment epithelium, may be involved in normal phagocytosis of the retinal outer segment, in infectious and ischemic processes, and in the pathogenesis of diabetic retinopathy. Nitric oxide contributes to basal tone in the retinal circulation. To date, findings are conflicting with respect to its role in retinal autoregulation. During glucose and oxygen deprivation, nitric oxide may increase blood flow and prevent platelet aggregation, but it may also mediate the toxic effects of excitatory amino acid release. This reactive, short-lived gas is involved in diverse processes within the retina, and its significance continues to be actively studied.

Topics: Animals; Choroid; Cyclic GMP; Cytokines; Humans; Hyperemia; Ischemia; Nitric Oxide; Nitric Oxide Synthase; Photoreceptor Cells; Pigment Epithelium of Eye; Rabbits; Rats; Retina; Retinal Diseases; Retinal Vessels; Vision, Ocular

The reason why patients with growth hormone (GH) deficiency (GHD) are at increased risk for premature cardiovascular death is still unclear. Although a variety of vascular risk factors have been identified in GHD, little is known regarding vascular reactivity and its contribution to premature arteriosclerosis.. We assessed vascular function in 7 childhood-onset, GH-deficient nontreated patients (age 22+/-3 years, body mass index [BMI] 25+/-1 kg/m(2)) and 10 healthy subjects (age 24+/-0.4 years, BMI 22+/-1 kg/m(2)) by using strain gauge plethysmography to measure forearm blood flow in response to vasodilatory agents. The increase in forearm blood flow to intrabrachial infusion of the endothelium-dependent vasodilator acetylcholine was significantly lower in GH-deficient nontreated patients than in control subjects (P:<0.05). Likewise, forearm release of nitrite and cGMP during acetylcholine stimulation was reduced in GH-deficient nontreated patients (P:<0.05 and P:<0.002 versus controls). The response to the endothelium-independent vasodilator sodium nitroprusside was also markedly blunted in GH-deficient patients compared with control subjects (P:<0.005). To confirm that abnormal vascular reactivity was due to GHD, we also studied 8 patients with childhood-onset GHD (age 31+/-2 years, BMI 24+/-1 kg/m(2)) who were receiving stable GH replacement therapy. In these patients, the response to both endothelium-dependent and -independent vasodilators, as well as forearm nitrite and cGMP, release was not different from that observed in normal subjects. Peak hyperemic response to 5-minute forearm ischemia was significantly reduced in GH-deficient nontreated patients (17.2+/-2.6 mL x dL(-1) x min(-1), P:<0.01) but not in GH-treated patients (24.8+/-3.3 mL x dL(-1) x min(-1)) compared with normal subjects (29.5+/-3.2 mL x dL(-1) x min(-1)).. The data support the concept that GH plays an important role in the maintenance of a normal vascular function in humans.

Topics: Acetylcholine; Adult; Blood Flow Velocity; Blood Pressure; Blood Vessels; Cyclic GMP; Dose-Response Relationship, Drug; Female; Forearm; Growth Hormone; Hormone Replacement Therapy; Humans; Ischemia; Male; Nitrites; Nitroprusside; Vasodilator Agents

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia recapitulates many characteristics of preeclampsia including maternal hypertension, intrauterine growth restriction (IUGR), and increased cytolytic natural killer cells (cNKs). While we have previously shown a 5-fold higher cytotoxicity of RUPP NKs versus normal pregnant NKs, their role in RUPP pathophysiology remains unclear. In this study, we tested the hypotheses that (1) adoptive transfer of RUPP-stimulated NKs will induce maternal hypertension and IUGR in normal pregnant control (Sham) rats and (2) adoptive transfer of Sham NKs will attenuate maternal hypertension and IUGR in RUPP rats.. On gestation day (GD)14, vehicle or 5 × 10. Adoptive transfer of RUPP NKs into Sham rats resulted in elevated NK activation, UARI, placental oxidative stress, and preproendothelin expression as well as reduced circulating nitrate/nitrite. This led to maternal hypertension and IUGR. RUPP recipients of Sham NKs demonstrated normalized NK activation, sFlt-1, circulating and placental VEGF, and UARI, which led to improved maternal blood pressure and normal fetal growth.. These data suggest a direct role for cNKs in causing preeclampsia pathophysiology and a role for normal NKs to improve maternal outcomes and IUGR during late gestation.

Topics: Adoptive Transfer; Animals; Cyclic GMP; Cytokines; Female; Fetal Growth Retardation; Ischemia; Killer Cells, Natural; Nitrates; Nitric Oxide Synthase Type III; Nitrites; Oxidative Stress; Phenotype; Placenta; Pre-Eclampsia; Pregnancy; Rats, Sprague-Dawley; Reactive Oxygen Species; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Rises in local neural activity trigger local increases of cerebral blood flow, which is essential to match local energy demands. However, the specific location of microvascular flow control is incompletely understood. Here, we used two-photon microscopy to observe brain microvasculature in vivo. Small spatial movement of a three-dimensional (3D) vasculature makes it challenging to precisely measure vessel diameter at a single x-y plane. To overcome this problem, we carried out four-dimensional (x-y-z-t) imaging of brain microvessels during exposure to vasoactive molecules in order to constrain the impact of brain movements on the recordings. We demonstrate that rises in synaptic activity, acetylcholine, nitric oxide, cyclic guanosine monophosphate, ATP-sensitive potassium channels, and endothelin-1 exert far greater effects on brain precapillary sphincters and first-order capillaries than on penetrating arterioles or downstream capillaries, but with similar kinetics. The high level of responsiveness at precapillary sphincters and first-order capillaries was matched by a higher level of α-smooth muscle actin in pericytes as compared to penetrating arterioles and downstream capillaries. Mathematical modeling based on 3D vasculature reconstruction showed that precapillary sphincters predominantly regulate capillary blood flow and pressure as compared to penetrating arterioles and downstream capillaries. Our results confirm a key role for precapillary sphincters and pericytes on first-order capillaries as sensors and effectors of endothelium- or brain-derived vascular signals.

Topics: Acetylcholine; Animals; Brain; Capillaries; Cyclic GMP; Endothelin-1; Endothelium, Vascular; Ion Channel Gating; Ischemia; KATP Channels; Mice; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Perfusion; Pericytes; Pressure; Receptors, Endothelin; S-Nitroso-N-Acetylpenicillamine; Vasodilation

Revascularization strategies and gene therapy for treatment of ischemic diseases remain to be fully optimized for use in human and veterinary clinical medicine. The continued evolution of such strategies must take into consideration two compounds, which act as critical effectors of angiogenesis by endothelial cells. Nevertheless, the nature of interaction between hydrogen sulfide (H2S) and nitric oxide (NO) remained undefined at the time of this writing.. The present study uses ZYZ-803, a novel synthetic H2S-NO hybrid molecule, which, under physiological conditions, slowly decomposes to release H2S and NO. This is observed to dose dependently mediate cell proliferation, migration, and tube-like structure formation in vitro along with increased angiogenesis in rat aortic rings, Matrigel plug in vivo, and a murine ischemic hind limb model. The effects of ZYZ-803 exhibited significantly greater potency than those of H2S and/or NO donor alone. The compound stimulated cystathionine γ-lyase (CSE) expression and endothelial NO synthase (eNOS) activity to produce H2S and NO. Blocking CSE and/or eNOS suppressed both H2S and NO generation as well as the proangiogenic effect of ZYZ-803. Sirtuin-1 (SIRT1), CSE, and/or eNOS small interfering RNA (siRNA) suppressed the angiogenic effect of ZYZ-803-induced SIRT1 expression, VEGF, and cyclic guanosine 5'-monophosphate (cGMP) levels. These gasotransmitters cooperatively regulated angiogenesis through an SIRT1/VEGF/cGMP pathway.. H2S and NO exert mutual influence on biological functions mediated by both compounds. Functional convergence occurs in the SIRT1-dependent proangiogenic processes. These two gasotransmitters are mutually required for physiological regulation of endothelial homeostasis. These ongoing characterizations of mechanisms by which ZYZ-803 influences angiogenesis provide expanding insight into strategies for treatment of ischemic diseases. Antioxid. Redox Signal. 25, 498-514.

Topics: Angiogenesis Inducing Agents; Animals; Cyclic GMP; Cystathionine gamma-Lyase; Delayed-Action Preparations; Drug Liberation; Endothelial Cells; Extremities; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen Sulfide; Ischemia; Male; Mice; Models, Biological; Molecular Structure; Nitric Oxide; Nitric Oxide Synthase Type III; Rats; Signal Transduction; Sirtuin 1; Vascular Endothelial Growth Factor A

Nitric oxide (NO) acts as essential regulator of vasculogenesis and angiogenesis and is critical for arteriogenesis. Whether NO's effects in vivo are mediated through NO-sensitive guanylyl cyclase (NO-GC) and thus by cGMP-dependent mechanisms has been only poorly addressed. Mice lacking NO-GC globally or specifically in smooth muscle cells (SMC) or endothelial cells (EC) were subjected to two established models for arteriogenesis and angiogenesis, namely hindlimb ischemia and oxygen-induced retinopathy. Our data clearly show the involvement of NO-GC in the recovery of blood flow after hindlimb ischemia, and this effect could be attributed to NO-GC in SMC. In the retina, global deletion of NO-GC led to reduced oxygen-induced vessel loss and hypoxia-induced capillary regrowth, whereas pathological neovascularization was increased. These effects were also seen in mice with SMC-specific NO-GC deletion but not in animals lacking NO-GC in EC. Intriguingly, NO-GC was found to be strongly expressed in retinal pericytes. Our data prove the involvement of NO-GC in growth and plasticity of hindlimb and retinal vasculature after ischemic/hypoxic insult.

Topics: Animals; Cyclic GMP; Endothelial Cells; Exons; Guanylate Cyclase; Hindlimb; Hypoxia; Image Processing, Computer-Assisted; Immunohistochemistry; Ischemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Smooth Muscle; Neovascularization, Pathologic; Nitric Oxide; Oxygen; Pericytes; Receptors, Cytoplasmic and Nuclear; Retina; Retinal Diseases; Signal Transduction; Soluble Guanylyl Cyclase; Time Factors

The kidney is vulnerable to hypoxia, and substantial efforts have been made to ameliorate renal ischemic injury secondary to pathological conditions. Stimulation of the nitrate-nitrite-nitric oxide pathway is associated with renal and cardiovascular protection in disease models, but less is known about the vascular effects during renal ischemia. This study was aimed at investigating the vascular effects of nitrite in the kidney during normoxic and ischemic conditions. Using a multiwire myograph system, we assessed nitrite-mediated relaxation (10(-9)-10(-4)mol/L) in isolated and preconstricted renal interlobar arteries from C57BL/6 mice under normal conditions (pO2 13kPa; pH 7.4) and with low oxygen tension and low pH to mimic ischemia (pO2 3kPa; pH 6.6). Xanthine oxidoreductase expression was analyzed by quantitative PCR, and production of reactive nitrogen species was measured by DAF-FM DA fluorescence. During normoxia significant vasodilatation (15±3%) was observed only at the highest concentration of nitrite, which was dependent on NO-sGC-cGMP signaling. The vasodilatory responses to nitrite were greatly sensitized and enhanced during hypoxia with low pH, demonstrating significant dilatation (11±1%) already in the physiological range (10(-8)mol/L), with a maximum response of 27±2% at 10(-4) mol/L. In contrast to normoxia, and to that observed with a classical NO donor (DEA NONOate), this sensitization was independent of sGC-cGMP signaling. Moreover, inhibition of various enzymatic systems reported to reduce nitrite in other vascular beds, i.e., aldehyde oxidase (raloxifene), aldehyde dehydrogenase (cyanamide), and NO synthase (L-NAME), had no effect on the nitrite response. However, inhibition of xanthine oxidoreductase (XOR; febuxostat or allopurinol) abolished the sensitized response to nitrite during hypoxia and acidosis. In conclusion, in contrast to normoxia, nitrite exerted potent vasorelaxation during ischemic conditions already at physiological concentrations. This effect was dependent on functional XOR but independent of classical downstream signaling by sGC-cGMP.

Topics: Animals; Cell Hypoxia; Cyclic GMP; Ischemia; Kidney; Male; Mice, Inbred C57BL; Nitrites; Second Messenger Systems; Vasodilation; Xanthine Oxidase

Preeclampsia is a complication of pregnancy that is marked by hypertension, proteinuria, and maternal endothelial dysfunction. A central factor in the etiology of the disease is the development of placental hypoxia/ischemia, which releases pathogenic soluble factors. There is currently no effective treatment for preeclampsia, but the phosphodiesterase-5 (PDE-5) inhibitor sildenafil has been suggested, as PDE-5 is enriched in the uterus, and its antagonism could improve uteroplacental function. Here, we report in the reduced uterine perfusion pressure (RUPP) rat model that administration of oral sildenafil is effective in attenuating placental ischemia-induced hypertension during gestation. RUPP animals have significantly elevated arterial pressure compared with control animals (132 ± 3 vs. 100 ± 2 mmHg; P < 0.05). Administration of oral sildenafil (45 mg·kg⁻¹·day⁻¹) had no effect on blood pressure in control rats but decreased pressure in RUPP rats (115 ± 1 mmHg; P < 0.05). RUPP induced changes in placental sFlt-1, and vascular endothelial growth factor (VEGF) was unaffected by sildenafil administration, as was the decrease in free plasma VEGF. RUPP animals had a significant increase in medullary PDE-5/β-actin ratio (1 ± 0.14 vs. 1.63 ± 0.18; P < 0.05) expression with a resulting reduction in renal medullary cGMP (1.5 ± 0.15 vs. 0.99 ± 0.1 pmol/μg protein, P < 0.05) compared with controls. Although sildenafil had no effect on renal medullary cGMP in control animals, it significantly increased cGMP in RUPP animals (1.3 ± 0.1 pmol/μg protein; P < 0.05). These data suggest that sildenafil might provide an effective therapeutic option for the management of hypertension during preeclampsia.

Topics: Actins; Administration, Oral; Animals; Antihypertensive Agents; Arterial Pressure; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Disease Models, Animal; Female; Ischemia; Kidney Medulla; Phosphodiesterase 5 Inhibitors; Piperazines; Placenta; Placental Circulation; Pre-Eclampsia; Pregnancy; Purines; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Sulfones; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vasodilator Agents

Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs.. We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx-derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated β-galactosidase activity. Mdx-derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell-derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density.. These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Topics: Animals; Apoptosis; Aspirin; Carcinoma, Lewis Lung; Caveolin 1; Cell Movement; Cell Proliferation; Cells, Cultured; Cellular Senescence; Coculture Techniques; Corneal Neovascularization; Cyclic GMP; Disease Models, Animal; Dystrophin; Endothelial Cells; Endothelium, Vascular; Ischemia; Mice; Mice, Inbred mdx; Muscular Dystrophy, Duchenne; Mutation; Myoblasts, Skeletal; Neovascularization, Pathologic; Neovascularization, Physiologic; Nitric Oxide; Nitric Oxide Synthase Type III; Time Factors

Type 2 diabetes is a key risk factor for ischemia-dependent pathology; therefore, a significant medical need exists to develop novel therapies that increase the formation of new vessels. We explored the therapeutic potential of epidermal growth factor receptor tyrosine kinase (EGFRtk) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibition in impaired ischemia-induced neovascularization in type 2 diabetes. Unilateral femoral artery ligation was performed in diabetic (db(-)/db(-)) and their control (db(-)/db(+)) mice for 4 weeks, followed by treatments with EGFRtk and ERK1/2 inhibitors (AG1478, 10 mg/kg/day and U0126, 400 μg/kg/day, respectively) for 3 weeks. Neovascularization, blood flow recovery, vascular and capillary density, and endothelial nitric oxide synthase activity were significantly impaired and were associated with enhanced EGFRtk and ERK1/2 activity in db(-)/db(-) mice. EGFRtk and ERK1/2 inhibitors did not have any effect in control mice, while in db(-)/db(-) mice there was a significant increase in neovascularization, blood flow recovery, vascular and capillary density, endothelial nitric oxide synthase activity, and were associated with a decrease in EGFRtk and ERK1/2 activity. Our data demonstrated that the inhibition of EGFRtk and ERK1/2 restored ischemia-induced neovascularization and blood flow recovery in type 2 diabetic mice. Thus, EGFRtk and ERK1/2 could be possible targets to protect from ischemia-induced vascular pathology in type 2 diabetes.

Topics: Animals; Blood Flow Velocity; Blood Glucose; Body Weight; Capillaries; Cyclic GMP; Diabetes Mellitus, Type 2; Diabetic Angiopathies; ErbB Receptors; Hindlimb; Insulin; Ischemia; Male; Mice; Nitric Oxide Synthase Type III; Phosphorylation; Receptor Protein-Tyrosine Kinases; RNA, Messenger; Vascular Endothelial Growth Factor A

This study examined the cytoprotective mechanisms of a combination of ischemic preconditioning (IPC) and allopurinol against liver injury caused by ischemia/reperfusion (I/R). Allopurinol (50mg/kg) was intraperitoneally administered 18 and 1h before sustained ischemia. A rat liver was preconditioned by 10 min of ischemia, followed by 10 min of reperfusion, and then subjected to 90 min of ischemia, followed by 5h of reperfusion. Rats were pretreated with adenosine deaminase (ADA), 3,7-dimethyl-1-[2-propargyl]-xanthine (DMPX), and N-nitro-l-arginine methyl ester (l-NAME) before IPC. Hepatic nitrite and nitrate and eNOS protein expression levels were increased by the combination of IPC and allopurinol. This increase was attenuated by ADA, DMPX, and l-NAME. I/R induced an increase in alanine aminotransferase activity, whereas it decreased the hepatic glutathione level. A combination of IPC and allopurinol attenuated these changes, which were abolished by ADA, DMPX, and l-NAME. The increase in the liver wet weight-to-dry weight ratio after I/R was attenuated by the combination of IPC and allopurinol. In contrast, hepatic bile flow was decreased after I/R, which was attenuated by the combination of IPC and allopurinol. These changes were restored by l-NAME. I/R induced a decrease in the level of mitochondrial dehydrogenase, whereas it increased mitochondrial swelling. A combination of IPC and allopurinol attenuated these changes, which were restored by ADA, DMPX, and l-NAME. Our findings suggest that a combination of IPC and allopurinol reduces post-ischemic hepatic injury by enhancing NO generation.

Topics: Adenosine Deaminase; Alanine Transaminase; Allopurinol; Animals; Arginine; Cyclic GMP; Glutathione; Ischemia; Ischemic Preconditioning; Liver; Male; Mitochondria, Liver; Mitochondrial Swelling; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Organ Size; Rats; Rats, Sprague-Dawley; Theobromine

Revascularization is an adaptive repair mechanism that restores blood flow to undersupplied ischemic tissue. Nitric oxide plays an important role in this process. Whether dietary nitrate, serially reduced to nitrite by commensal bacteria in the oral cavity and subsequently to nitric oxide and other nitrogen oxides, enhances ischemia-induced remodeling of the vascular network is not known.. Mice were treated with either nitrate (1 g/L sodium nitrate in drinking water) or sodium chloride (control) for 14 days. At day 7, unilateral hind-limb surgery with excision of the left femoral artery was conducted. Blood flow was determined by laser Doppler. Capillary density, myoblast apoptosis, mobilization of CD34(+)/Flk-1(+), migration of bone marrow-derived CD31(+)/CD45(-), plasma S-nitrosothiols, nitrite, and skeletal tissue cGMP levels were assessed. Enhanced green fluorescence protein transgenic mice were used for bone marrow transplantation. Dietary nitrate increased plasma S-nitrosothiols and nitrite, enhanced revascularization, increased mobilization of CD34(+)/Flk-1(+) and migration of bone marrow-derived CD31(+)/CD45(-) cells to the site of ischemia, and attenuated apoptosis of potentially regenerative myoblasts in chronically ischemic tissue. The regenerative effects of nitrate treatment were abolished by eradication of the nitrate-reducing bacteria in the oral cavity through the use of an antiseptic mouthwash.. Long-term dietary nitrate supplementation may represent a novel nutrition-based strategy to enhance ischemia-induced revascularization.

Topics: Animal Feed; Animals; Bone Marrow Transplantation; Cell Movement; Chronic Disease; Cyclic GMP; Dietary Supplements; Disease Models, Animal; Femoral Artery; Green Fluorescent Proteins; Hindlimb; Ischemia; Laser-Doppler Flowmetry; Ligation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myoblasts; Nitrates; Nitric Oxide; Nitrites; Regeneration; Regional Blood Flow; S-Nitrosothiols

Arterial hypertension is a major risk factor that can lead to complication of peripheral vascular disease due, in part, to endothelial dysfunction. Because sodium nitrite (SN) can be converted to nitric oxide (NO), which counteracts endothelial dysfunction, we explored the effect of nitrite on neovascularization following hind limb ischemia in different models of hypertension (HT). Chronic delivery of angiotensin II (Ang II, 400 ng/kg/min) or N(omega)-nitro-L-arginine-methyl-ester (L-NAME, 0.1 g/L) was used for a 2-week period to induce hypertension. Mice were subjected to femoral artery ligation-induced ischemia in the hind limb followed by treatment with SN (50 mg/L) for 2 weeks. SN significantly reduced systolic arterial blood pressure in mice receiving Ang II and L-NAME but had no effect in sham animals. After 2 weeks, blood flow and microangiography showed 60 % ± 1.0 recovery in sham compared with 40 % ± 1.3 in HT mice. Importantly, sham and HT mice treated with SN showed a 100 % blood flow recovery associated with normalization in capillary density. The inhibition of xanthine-oxido-reductase (allopurinol) or VEGFR (SU-5416) prevented the neovascularization in HT mice treated with SN. Cyclic GMP (cGMP) content in the hind limb was significantly increased in mice treated with SN compared with non-treated mice. Nitrite/nitrate content was only increased in the sham group treated with SN. Immunoprecipitation and Western blot analysis revealed an increase in eNOS/Akt/VEGFR phosphorylation in skeletal muscle from mice treated with SN compared with non-treated mice. Our findings indicate that SN therapy rescues the neovascularization and blood flow recovery in the ischemic hind limb of sham and HT mice likely through the Akt/NO/cGMP and VEGFR pathways.

Topics: Allopurinol; Angiotensin II; Animals; Arterial Pressure; Capillaries; Cyclic AMP; Cyclic GMP; Femoral Artery; Hindlimb; Hypertension; Indoles; Ischemia; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Neovascularization, Pathologic; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Proto-Oncogene Proteins c-akt; Pyrroles; Receptors, Vascular Endothelial Growth Factor; Regional Blood Flow; Sodium Nitrite; Xanthine Dehydrogenase

Circulating angiogenic cells (CACs), represent a potential new therapeutic tool for the treatment of cardiovascular diseases, but their regenerative function is impaired in patients with coronary artery disease (CAD) and cardiac risk factors. The objective of this study is to assess the effect of lentiviral overexpression of endothelial nitric oxide synthase (eNOS) on the activity of CACs from patients with CAD and cardiac risk factors. In vitro and in vivo assays were employed to evaluate the regenerative capacity of the cells compared to CACs derived from healthy volunteers. Lentiviral eNOS transduction of cells from CAD patients significantly improved chemotactic migration compared with sham transduction, and increased the ability of CACs to induce angiogenic tube formation when cocultured with human umbilical vein endothelial cells (HUVECs) on Matrigel. In addition, eNOS transduction restored the ability of patient-derived CACs to enhance neovascularization and improve ischemic hind limb perfusion, approaching the efficacy of cells from healthy donors. These data indicate that CAC dysfunction seen in high-risk patients can be partially reversed by eNOS overexpression, suggesting that ex vivo gene delivery may improve the efficacy of autologous cell therapy for cardiovascular disease.

Topics: Adult; Animals; Cell Movement; Cells, Cultured; Coronary Artery Disease; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Extremities; Female; Humans; Ischemia; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Nitric Oxide; Nitric Oxide Synthase Type III; Stem Cell Transplantation

Neovascularization is a physiologic repair process that partly depends on nitric oxide. Extracellular superoxide dismutase (EcSOD) is the major scavenger of superoxide. It is an important regulator of nitric oxide bioavailability and thus protects against vascular dysfunction. We hypothesized that overexpression of EcSOD in skeletal muscle would improve recovery from hind-limb ischemia.. Adeno-associated virus serotype 9 (AAV9) vectors expressing EcSOD or luciferase (control) from the cytomegalovirus promoter were cross-packaged into AAV9 capsids and injected intramuscularly into the hind-limb muscles (1 × 10(11) viral genomes/limb) of 12-week-old mice. Ischemia was induced after intramuscular injections. Laser Doppler was used to measure limb perfusion on days 0, 7, and 14 after injection. Values were expressed as a ratio relative to the nonischemic limb. EcSOD expression was measured by Western blotting. Capillary density was documented by immunohistochemical staining for platelet endothelial cell adhesion molecule. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated biotin-deoxy uridine triphosphate nick-end labeling and necrosis was visually evaluated daily.. EcSOD expression was twofold upregulated in EcSOD treated vs control ischemic muscles at day 14. Capillary density (capillaries/fiber) was 1.9-fold higher in treated (1.65 ± 0.02) vs control muscle (0.78 ± 0.17, P < .05). Recovery of perfusion ratio at day 14 after ischemia was 1.5-fold greater in EcSOD vs control mice (P < .05). The percentage of apoptotic nuclei was 1.3% ± 0.4% in EcSOD-treated mice compared with 4.2% ± 0.2% in controls (P < .001). Limb necrosis was also significantly lower in EcSOD vs control mice.. AAV9-mediated overexpression of EcSOD in skeletal muscle significantly improves recovery from hind-limb ischemia in mice, consistent with improved capillary density and perfusion ratios in treated mice.

Topics: Animals; Apoptosis; Blotting, Western; Capillaries; Cyclic GMP; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Hindlimb; Immunohistochemistry; In Situ Nick-End Labeling; Injections, Intramuscular; Ischemia; Laser-Doppler Flowmetry; Luciferases, Firefly; Male; Mice; Mice, Inbred BALB C; Muscle, Skeletal; Necrosis; Neovascularization, Physiologic; Platelet Endothelial Cell Adhesion Molecule-1; Recombinant Fusion Proteins; Recovery of Function; Regional Blood Flow; Superoxide Dismutase; Time Factors

BACKGROUND- Reactive oxygen species serve signaling functions in the vasculature, and hypoxia has been associated with increased reactive oxygen species production. NADPH oxidase 4 (Nox4) is a reactive oxygen species-producing enzyme that is highly expressed in the endothelium, yet its specific role is unknown. We sought to determine the role of Nox4 in the endothelial response to hypoxia.. Hypoxia induced Nox4 expression both in vitro and in vivo and overexpression of Nox4 was sufficient to promote endothelial proliferation, migration, and tube formation. To determine the in vivo relevance of our observations, we generated transgenic mice with endothelial-specific Nox4 overexpression using the vascular endothelial cadherin promoter (VECad-Nox4 mice). In vivo, the VECad-Nox4 mice had accelerated recovery from hindlimb ischemia and enhanced aortic capillary sprouting. Because endothelial nitric oxide synthase (eNOS) is involved in endothelial angiogenic responses and eNOS is activated by reactive oxygen species, we probed the effect of Nox4 on eNOS. In cultured endothelial cells overexpressing Nox4, we observed a significant increase in eNOS protein expression and activity. To causally address the link between eNOS and Nox4, we crossed our transgenic Nox4 mice with eNOS(-/-) mice. Aortas from these mice did not demonstrate enhanced aortic sprouting, and VECad-Nox4 mice on the eNOS(-/-) background did not demonstrate enhanced recovery from hindlimb ischemia.. Collectively, we demonstrate that augmented endothelial Nox4 expression promotes angiogenesis and recovery from hypoxia in an eNOS-dependent manner.

Topics: Animals; Cattle; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Endothelial Cells; Enzyme Induction; Genetic Therapy; Genetic Vectors; Hindlimb; Humans; Ischemia; Mice; Mice, Transgenic; NADPH Oxidase 4; NADPH Oxidases; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Reactive Oxygen Species; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering

We examined whether phosphodiesterase-5 (PDE5) inhibition can promote ischemia-induced angiogenesis.. Unilateral hindlimb ischemia was generated by resecting right femoral artery in wild-type C3H/He mice, treated with either vehicle or a PDE5 inhibitor vardenafil (10 mg/kg per day). Four weeks after surgery, vardenafil significantly enhanced blood flow recovery and augmented capillary collateral formation in ischemic muscle (blood flow ratios of ischemic/nonischemic leg: 0.52+/-0.17 [vehicle] versus 0.92+/-0.09 [vardenafil], P<0.01). Vardenafil upregulated protein expression of vascular endothelial growth factor and hypoxia-inducible factor (HIF)-1 alpha in ischemic muscle and enhanced mobilization of Sca-1/Flk-1-positive endothelial progenitor cells (EPCs) in peripheral blood and bone marrow, contributing to neovascularization. Vardenafil also promoted capillary-like tube formation of human umbilical vein endothelial cells and increased the number of human blood mononuclear cell-derived EPCs in vitro. Furthermore, reporter assays showed that vardenafil and cGMP activated the transactivation activity of HIF-1 under hypoxia. These effects of vardenafil were markedly inhibited by genetic ablation of endothelial nitric oxide synthase, a soluble guanylate cyclase inhibitor, and a protein kinase G inhibitor, respectively.. Our results suggest that PDE5 inhibition enhances ischemia-induced angiogenesis with mobilization of EPCs through a protein kinase G-dependent HIF-1/vascular endothelial growth factor pathway. PDE5 inhibition may have a therapeutic potential to treat ischemic cardiovascular diseases.

Topics: Angiogenesis Inducing Agents; Animals; Capillaries; Cell Hypoxia; Cell Movement; Cells, Cultured; Collateral Circulation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Disease Models, Animal; Endothelial Cells; Green Fluorescent Proteins; Hindlimb; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Ischemia; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Muscle, Skeletal; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Piperazines; Recovery of Function; Regional Blood Flow; RNA Interference; Signal Transduction; Stem Cells; Sulfones; Time Factors; Transfection; Triazines; Vardenafil Dihydrochloride; Vascular Endothelial Growth Factor A

Topics: Acute Kidney Injury; Anti-Bacterial Agents; Biopsy; Cyclic GMP; Escherichia coli Infections; Histiocytes; Humans; Ischemia; Kidney; Kidney Diseases; Malacoplakia; Male; Middle Aged; Periodic Acid-Schiff Reaction; Postoperative Complications; Pyelonephritis; Transplants; Urinary Tract Infections

Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPalpha (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.

Topics: Animals; CD47 Antigen; Cells, Cultured; Cyclic GMP; Humans; Ischemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Protein Binding; Receptors, Immunologic; Signal Transduction; Thrombospondins

Tissue ischemia and ischemia-reperfusion (I/R) remain sources of cell and tissue death. Inability to restore blood flow and limit reperfusion injury represents a challenge in surgical tissue repair and transplantation. Nitric oxide (NO) is a central regulator of blood flow, reperfusion signaling and angiogenesis. De novo NO synthesis requires oxygen and is limited in ischemic vascular territories. Nitrite (NO(2-)) has been discovered to convert to NO via heme-based reduction during hypoxia, providing a NO synthase independent and oxygen-independent NO source. Furthermore, blockade of the matrix protein thrombospondin-1 (TSP1) or its receptor CD47 has been shown to promote downstream NO signaling via soluble guanylate cyclase (sGC) and cGMP-dependant kinase. We hypothesized that nitrite would provide an ischemic NO source that could be potentiated by TSP1-CD47 blockade enhancing ischemic tissue survival, blood flow and angiogenesis. Both low dose nitrite and direct blockade of TSP1-CD47 interaction using antibodies or gene silencing increased acute blood flow and late tissue survival in ischemic full thickness flaps. Nitrite and TSP1 blockade both enhanced in vitro and in vivo angiogenic responses. The nitrite effect could be abolished by inhibition of sGC and cGMP signaling. Potential therapeutic synergy was tested in a more severe ischemic flap model. We found that combined therapy with nitrite and TSP1-CD47 blockade enhanced flap perfusion, survival and angiogenesis to a greater extent than either agent alone, providing approximately 100% flap survival. These data provide a new therapeutic paradigm for hypoxic NO signaling through enhanced cGMP mediated by TSP1-CD47 blockade and nitrite delivery.

Topics: Analysis of Variance; Animals; Back; CD47 Antigen; Cyclic GMP; Enzyme Inhibitors; Gene Silencing; Ischemia; Male; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Nitric Oxide; Nitrites; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Signal Transduction; Surgical Flaps; Thrombospondin 1; Xanthine Dehydrogenase

Ischemia-reperfusion causes endothelial dysfunction. Prolongation of acidosis during initial cardiac reperfusion limits infarct size in animal models, but the effects of acidic reperfusion on vascular function are unknown. The present work analyzes the effects of acidic reoxygenation on vascular responses to different agonists in rat aortic rings. Arterial rings obtained from Sprague-Dawley rat aorta were placed in organ baths containing a Krebs solution oxygenated at 37 degrees C (pH 7.4). After equilibration (30 mN, 1 h), the effects of acidosis (pH 6.4) on aortic responses to acetylcholine and norepinephrine were initially assessed under normoxic conditions. Thereafter, the effects of acidosis during hypoxia (1 h) or reoxygenation on aortic responses to acetylcholine, norepinephrine, or sodium nitroprusside were analyzed and compared with those observed in control rings. Acidosis did not modify aortic responses to acetylcholine or adrenaline during normoxia. In contrast, rings submitted to hypoxia and reoxygenated at pH 7.4 showed a reduction in vasodilator responses to acetylcholine and in contractions to norepinephrine with no change in responses to sodium nitroprusside. Reoxygenation at pH 6.4 did not modify the depressed response to norepinephrine but enhanced the recovery of acetylcholine-induced vasorelaxation. Cumulative concentration-response curves to acetylcholine showed an increased responsiveness to this drug in rings reoxygenated at a low pH. This functional improvement was associated with the preservation of aortic cGMP content after stimulation of reoxygenated rings with acetylcholine. In conclusion, acidic reoxygenation preserves endothelial function in arterial rings submitted to simulated ischemia, likely through the preservation of cGMP signaling.

Topics: Acetylcholine; Acidosis; Animals; Aorta, Thoracic; Cell Death; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Ischemia; Male; Nitroprusside; Norepinephrine; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

To investigate whether nitric oxide (NO) and/or cGMP protects the retina from chemical ischemia and underlie somatostatin's neuroprotective effects.. Eyecups of female Sprague-Dawley rats were incubated with PBS or the chemical ischemia mixture [iodoacetic acid (5 mM)/sodium cyanate (25 mM)] in the absence or presence of (1) arginine (0.05-2.0 mM), the substrate of nitric oxide synthase (NOS); (2) the NO donors sodium nitroprusside (SNP; 0.25-4.0 mM), 3-morpholinosydnonimine (SIN-1; 0.1, 0.3, 1.0 mM), SIN-1 (0.1 mM)/L-cysteine (5 mM, peroxynitrite scavenger), and NONOate (1, 5, 10 microM, slow NO releaser); (3) 8-Br-cGMP (0.1, 0.5, 1.0 mM); (4) BIM23014 (sst(2) receptor agonist; 1 microM), alone or in the presence of (5) the NOS inhibitor N(gamma)-monomethyl-L-arginine (NMMA; 0.5 mM); or (6) the guanylyl cyclase inhibitors 1H-[1,2,4]oxadiazolol [4,3-a]quinoxalin-1-one (ODQ;100 microM) and NS2028 (50 microM) for 60 minutes, at 5%CO(2)/air in 37 degrees C. The effect of SIN-1 (0.1, 0.3, 1.0, or 3.0 mM) on the retina was also examined. Subsequently, the eyecups were fixed and sectioned for choline acetyltransferase (ChAT) immunoreactivity and TUNEL staining.. Arginine and SNP had no effect on the chemical ischemia-induced toxicity. SIN-1, NONOate, and 8-Br-cGMP produced a concentration-dependent protective effect, as shown by ChAT immunoreactivity. TUNEL staining also confirmed the neuroprotective effect of these agents. L-cysteine partially reduced the SIN-1-induced protective effect. SIN-1 alone was toxic only at the highest concentration used (3 mM). NMMA, ODQ, and NS2028 reversed the protective effect of BIM23014.. The results suggest that a NO/peroxynitrite/cGMP mechanism may be important in the protection of the retina from ischemic insult. Furthermore, the NO/sGC/cGMP pathway is involved in the neuroprotective effects of sst(2) ligands against retinal ischemia.

Topics: Animals; Choline O-Acetyltransferase; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Fluorescent Antibody Technique, Indirect; In Situ Nick-End Labeling; Ischemia; Molsidomine; Nitric Oxide; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Retinal Diseases; Retinal Vessels; Somatostatin; Spermine

Since the generation of nitric oxide (NO) is an essential step in the trigger phase of ischemic preconditioning, short-term inhalation of NO before ischemia should ameliorate ischemia/reperfusion (I/R) injury of the lung. We tested this hypothesis in high oxygen (>99%) ventilated rats in order to additionally evaluate compatibility of NO and exposure to hyperoxia. Male adult Sprague-Dawley rats inhaled NO (15 ppm, 10 min) before the left lung hilum was clamped for 1 h, and the reperfusion phase was observed for 4 h (NO group). Animals in the I/R group underwent the same treatment, but without NO inhalation. A third group without I/R served as time-matched controls. Animals in the I/R group showed severe I/R injury in terms of arterial pO2 (apO2), which was reduced to 22% of surgical controls (SCs) at time point 30 min reperfusion, and increased endothelial permeability (Evans blue procedure). The pretreatment with NO attenuated these effects. The pO2 after 4 h reperfusion was still 3.0-fold higher in the NO group compared to I/R. In contrast, the I/R- and hyperoxia-induced invasion of leukocytes, as determined by measuring myeloperoxidase (MPO) activity, was not affected by NO. These data were correlated with the activity of major cellular signaling pathways by measuring the phosphorylation at activating and inhibitory sites of extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, protein kinase B (AKT), and glycogen synthase kinase 3beta (GSK-3beta), and by determination of cGMP in plasma and lung tissue. Inhalation of NO partly prevented the loss of activation by I/R and hyperoxic ventilation of ERK, JNK, and AKT, and it reduced the I/R-induced activation of GSK-3beta. The level of cGMP in plasma and lung tissue was increased in the NO group after 4 h reperfusion. In conclusion, application of inhaled NO in the preconditioning mode prevented I/R injury in the rat lung without interfering effects of hyperoxic ventilation. The effects of NO on cellular signaling pathways resemble mechanisms of ischemic preconditioning, but further studies have to evaluate the physiological relevance of these results.

Topics: Administration, Inhalation; Animals; Capillary Permeability; Cyclic GMP; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Glycogen Synthase Kinases; Hyperoxia; Ischemia; Ischemic Preconditioning; Lung; Male; MAP Kinase Signaling System; Nitric Oxide; Proto-Oncogene Proteins c-akt; Pulmonary Gas Exchange; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction

Cerebral microvascular endothelial cells form the anatomical basis of the blood-brain barrier (BBB), and the tight junctions of the BBB are critical for maintaining brain homeostasis and low permeability. Ischemia/reperfusion is known to damage the tight junctions of BBB and lead to permeability changes. Here we investigated the protective role of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), against chemical hypoxia and hypoxia/reoxygenation (H/R)-induced BBB hyperpermeability using adult rat brain endothelial cell culture (ARBEC). YC-1 significantly decreased CoCl2- and H/R-induced hyperpermeability of fluorescein isothiocyanate (FITC)-dextran in cell culture inserts. It was found that the decrease and disorganization of tight junction protein zonular occludens-1 (ZO-1) in response to CoCl2, and H/R was antagonized by YC-1. The protection of YC-1 may result from the inhibition of HIF-1alpha accumulation and production of its downstream target vascular endothelial growth factor (VEGF). VEGF alone significantly increased FITC-dextran permeability and down-regulated mRNA and protein levels of ZO-1 in ARBECs. We further used animal model to examine the effect of YC-1 on BBB permeability after cerebral ischemia/reperfusion. It was found that YC-1 significantly protected the BBB against ischemia/reperfusion-induced injury. Taken together, these results indicate that YC-1 may inhibit HIF-1alpha accumulation and VEGF production, which in turn protect BBB from injury caused by hypoxia.

Topics: Animals; Blood-Brain Barrier; Cell Hypoxia; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Ischemia; Male; Membrane Proteins; Permeability; Phosphoproteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Vascular Endothelial Growth Factor A; Zonula Occludens-1 Protein

Neovascularization is an important physiological repair mechanism in response to ischemic injury, and its process is dependent on reactive oxygen species (ROS). Overproduction of superoxide anion (O2-) rather contributes to various cardiovascular diseases. The extracellular superoxide dismutase (ecSOD) is one of the major antioxidant enzymes against O2- in blood vessels; however, its role in neovascularization induced by tissue ischemia is unknown. Here we show that hindlimb ischemia of mice stimulates a significant increase in ecSOD activity in ischemic tissues where ecSOD protein is highly expressed at arterioles. In mice lacking ecSOD, ischemia-induced increase in blood flow recovery, collateral vessel formation, and capillary density are significantly inhibited. Impaired neovascularization in ecSOD(-/-) mice is associated with enhanced O2- production, TUNEL-positive apoptotic cells and decreased levels of NO2-/NO3- and cGMP in ischemic tissues as compared with wild-type mice, and it is rescued by infusion of the SOD mimetic tempol. Recruitment of inflammatory cells into ischemic tissues as well as numbers of inflammatory cells and endothelial progenitor cells (c-kit+/CD31+ cells) in both peripheral blood and bone marrow (BM) are significantly reduced in these knockout mice. Of note, ecSOD expression is markedly increased in BM after ischemia. NO2-/NO3- and cGMP levels are decreased in ecSOD(-/-) BM. Transplantation of wild-type BM into ecSOD(-/-) mice rescues the defective neovascularization. Thus, ecSOD in BM and ischemic tissues induced by hindlimb ischemia may represent an important compensatory mechanism that blunts the overproduction of O2-, which may contribute to reparative neovascularization in response to ischemic injury.

Topics: Animals; Antioxidants; Apoptosis; Bone Marrow Cells; Bone Marrow Transplantation; Cell Differentiation; Cells, Cultured; Cyclic GMP; Cyclic N-Oxides; Endothelium, Vascular; Extracellular Space; Hindlimb; Ischemia; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neovascularization, Physiologic; Nitrates; Nitrites; Reactive Oxygen Species; Spin Labels; Stem Cells; Superoxide Dismutase; Vasculitis

Decreased blood flow secondary to peripheral vascular disease underlies a significant number of chronic diseases that account for the majority of morbidity and mortality among the elderly. Blood vessel diameter and blood flow are limited by the matricellular protein thrombospondin-1 (TSP1) through its ability to block responses to the endogenous vasodilator nitric oxide (NO). In this study we investigate the role TSP1 plays in regulating blood flow in the presence of advanced age and atherosclerotic vascular disease.. Mice lacking TSP1 or CD47 show minimal loss of their resistance to ischemic injury with age and increased preservation of tissue perfusion immediately after injury. Treatment of WT and apolipoprotein E-null mice using therapeutic agents that decrease CD47 or enhance NO levels reverses the deleterious effects of age- and diet-induced vasculopathy and results in significantly increased tissue survival in models of ischemia.. With increasing age and diet-induced atherosclerotic vascular disease, TSP1 and its receptor CD47 become more limiting for blood flow and tissue survival after ischemic injury. Drugs that limit TSP1/CD47 regulation of blood flow could improve outcomes from surgical interventions in the elderly and ameliorate vascular complications attendant to aging.

Topics: Aging; Animals; Apolipoproteins E; Atherosclerosis; Blood Flow Velocity; CD47 Antigen; Cell Survival; Collateral Circulation; Cyclic GMP; Disease Models, Animal; Femoral Artery; Hindlimb; Ischemia; Ligation; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Necrosis; Nitric Oxide; Peripheral Vascular Diseases; Regional Blood Flow; Signal Transduction; Thrombospondin 1; Vasodilation

Carbon monoxide (CO), an endogenous cytoprotective product of heme oxygenase type-1 regulates target thrombotic and inflammatory genes in ischemic stress. Regulation of the gene encoding early growth response 1 (Egr-1), a potent transcriptional activator of deleterious thrombotic and inflammatory cascades, may govern CO-mediated ischemic lung protection. The exact signaling mechanisms underlying CO-mediated cytoprotection are not well understood. In this study we tested the hypothesis that inhibition of mitogen-activated protein kinase-dependent Egr-1 expression may be pivotal in CO-mediated ischemic protection. In an in vivo isogeneic rat lung ischemic injury model, inhaled CO not only diminished fibrin accumulation and leukostasis and improved gas exchange and survival but also suppressed extracellular signal-regulated kinase (ERK) activation, Egr-1 expression, and Erg DNA-binding activity in lung tissue. Additionally, CO-mediated inhibition of Egr-1 reduced expression of target genes, such as tissue factor, serpine-1, interleukin-1, and TNF-alpha. However, CO failed to inhibit serpine-1 expression after unilateral lung ischemia in mice null for the Egr-1 gene. In RAW macrophages in vitro, hypoxia-induced Egr-1 mRNA expression was ERK-dependent, and CO-mediated suppression of ERK activation resulted in Egr-1 inhibition. Furthermore, CO suppression of ERK phosphorylation was reversed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but was insensitive to cAMP-dependent protein kinase A inhibition with H89 and NO synthase inhibition with l-nitroarginine methyl ester. This finding indicates that CO suppresses ERK in a cGMP-dependent but cAMP/protein kinase A- and NO-independent manner. Together, these data identify a unifying molecular mechanism by which CO interrupts proinflammatory and prothrombotic mediators of ischemic injury.

Topics: Animals; Carbon Monoxide; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Early Growth Response Protein 1; Gene Expression Regulation; Graft Survival; Hypoxia; Ischemia; Lung Diseases; Lung Transplantation; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Nitric Oxide; Rats

Carbon dioxide-rich water bathing has the effect of vasodilatation, whereas it remains undetermined whether this therapy exerts an angiogenic action associated with new vessel formation.. Unilateral hindlimb ischemia was induced by resecting the femoral arteries of C57BL/J mice. Lower limbs were immersed in CO2-enriched water (CO2 concentration, 1000 to 1200 mg/L) or freshwater (control) at 37 degrees C for 10 minutes once a day. Laser Doppler imaging revealed increased blood perfusion in ischemic limbs of CO2 bathing (38% increase at day 28, P<0.001), whereas N(G)-nitro-L-arginine methyl ester treatment abolished this effect. Angiography or immunohistochemistry revealed that collateral vessel formation and capillary densities were increased (4.1-fold and 3.7-fold, P<0.001, respectively). Plasma vascular endothelial growth factor (VEGF) levels were elevated at day 14 (18%, P<0.05). VEGF mRNA levels, phosphorylation of NO synthase, and cGMP accumulation in the CO2-bathed hindlimb muscles were increased (2.7-fold, 2.4-fold, and 3.4-fold, respectively) but not in forelimb muscles. The number of circulating Lin-/Flk-1+/CD34- endothelial-lineage progenitor cells was markedly increased by CO2 bathing (24-fold at day 14, P<0.001). The Lin-/Flk-1+/CD34- cells express other endothelial antigens (endoglin and VE-cadherin) and incorporated acetylated LDL.. Our present study demonstrates that CO2 bathing of ischemic hindlimb causes the induction of local VEGF synthesis, resulting in an NO-dependent neocapillary formation associated with mobilization of endothelial progenitor cells.

Topics: Animals; Baths; Carbon Dioxide; Collateral Circulation; Cyclic GMP; Endothelium, Vascular; Hematopoietic Stem Cell Mobilization; Hindlimb; Ischemia; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Nitric Oxide; Stem Cells; Vascular Endothelial Growth Factor A

The effects of aging on angiogenesis (vascular sprouting) and vasculogenesis (endothelial precursor cell [EPC] incorporation into vessels) are not well known. We examined whether ischemia-induced angiogenesis/vasculogenesis is altered in klotho (kl) mutant mice, an animal model of typical aging.. After unilateral hindlimb ischemia, laser Doppler blood-flow (LDBF) analysis revealed a decreased ischemic-normal LDBF ratio in kl mice. Tissue capillary density was also suppressed in kl mice (+/+>+/kl>kl/kl). Aortic-ring culture assay showed impaired angiogenesis in kl/kl mice, accompanied by reduced endothelium-derived nitric oxide release. Moreover, the rate of transplanted homologous bone marrow cells incorporated into capillaries in ischemic tissues (vasculogenesis) was lower in kl/kl mice than in wild-type (+/+) mice, which was associated with a decrease in the number of c-Kit+CD31+ EPC-like mononuclear cells in bone marrow and in peripheral blood. Finally, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor cerivastatin restored the impaired neovascularization in kl/kl mice, accompanied by an increase in c-Kit+CD31+ cells in bone marrow and peripheral blood, and enhanced angiogenesis in the aortic-ring culture.. Angiogenesis and vasculogenesis are impaired in kl mutant mice, a model of typical aging. Moreover, the age-associated impairment of neovascularization might be a new target of statin therapy.

Topics: Aging, Premature; Animals; Aorta, Thoracic; Bone Marrow Transplantation; Collateral Circulation; Cyclic GMP; Glucuronidase; Hindlimb; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ischemia; Klotho Proteins; Laser-Doppler Flowmetry; Membrane Proteins; Mice; Mice, Mutant Strains; Models, Animal; Muscle, Skeletal; Neovascularization, Pathologic; Nitrates; Nitric Oxide; Nitrites; Organ Culture Techniques; Pyridines; Ultrasonography

It remains undetermined whether continuous endothelial nitric oxide (NO) overexpression exerts angiogenic action. We surgically induced hindlimb ischemia in transgenic mice overexpressing endothelial NO synthase in the endothelium (eNOS-Tg) and studied neocapillary formation, ischemia-induced vascular endothelial growth factor (VEGF) expression, cGMP accumulation, and Akt/PKB signaling. Laser Doppler imaging revealed a markedly increased recovery of blood perfusion in ischemic limbs of eNOS-Tg mice (44% increase) compared with that in wild-type mice. Angiography showed a marked increase in basal and ischemia-induced collateral vessel formation in eNOS-Tg mice. Basal capillary densities and tissue cGMP levels were increased in eNOS-Tg mice (1.8-fold and 1.6-fold versus wild-type mice, respectively). Ischemia-induced neocapillary formation and cGMP accumulation were markedly increased in eNOS-Tg mice (3.6-fold and 4.1-fold versus preischemia levels, respectively), whereas those in wild-type mice were much less (1.8-fold and 1.5-fold, respectively). Basal and time-dependent VEGF expression in ischemic muscles did not differ between eNOS-Tg and wild-type mice. Basal and VEGF-mediated Akt phosphorylation in aortas was similar between eNOS-Tg and wild-type mice. Aortic basal eNOS expression was increased 3.3-fold, and VEGF-mediated eNOS phosphorylation was markedly induced in aortas of eNOS-Tg compared with preischemia levels (4.2-fold), whereas much smaller changes were observed in wild-type mice (1.8-fold increase). Our study demonstrates that overexpression of eNOS protein causes a marked increase in neocapillary formation in response to tissue ischemia without affecting ischemia-induced VEGF expression or VEGF-mediated Akt phosphorylation.

Topics: Angiography; Animals; Blood Circulation; Capillaries; Collateral Circulation; Cyclic GMP; Endothelial Growth Factors; Hindlimb; Intercellular Signaling Peptides and Proteins; Ischemia; Laser-Doppler Flowmetry; Lymphokines; Male; Mice; Mice, Transgenic; Neovascularization, Physiologic; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMPcGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPscGMPcGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk12 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPscGMPcGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia.

Topics: Animals; Atrial Natriuretic Factor; Blood Vessels; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Expression; Gene Transfer Techniques; Humans; Inflammation; Ischemia; Mice; Mice, Knockout; Mice, Transgenic; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Neovascularization, Physiologic; Regeneration

We studied the effect of chronic ischemia on prostatic smooth muscle contraction in the rabbit.. New Zealand male rabbits weighing 3 to 3.5 kg were assigned to 2 groups. Group 1 (10 rabbits) underwent balloon endothelial injury of the iliac arteries and received a 0.5% cholesterol diet for 4 weeks and then a regular diet for 8 weeks. Control group 2 (10 rabbits) received a regular diet. After 12 weeks the animals were anesthetized. Iliac artery and prostate blood flow was recorded. Prostate tissues were prepared for isometric tension measurement, enzyme immunoassay to determine cyclic guanosine monophosphate (cGMP) release and histological examination.. In group 1 atherosclerosis as well as a significant decrease in iliac artery and prostate blood flow were observed. Ischemia significantly increased prostatic tissue contraction, decreased cGMP release and led to capsular and stromal thickening, and epithelial atrophy. The alpha1-adrenoceptor blocker doxazosin and the phosphodiesterase-5 inhibitor sildenafil citrate significantly decreased the contraction of control and ischemic tissues. Doxazosin was more effective in decreasing contractions when it was combined with sildenafil or the nitric oxide (NO) precursor L-arginine. In contrast, doxazosin was less effective when it was combined with the NO synthase inhibitor N omega-nitro-L-arginine or with the guanylate cyclase inhibitor methylene blue. Doxazosin significantly increased cGMP release in control tissues but not in ischemic tissues. Sildenafil significantly increased cGMP release in control and ischemic tissues.. Ischemia increased prostatic smooth muscle contraction and led to marked structural damage. Stimulators of NO synthesis and cGMP production enhanced the efficacy of doxazosin in decreasing prostatic tissue contraction. Sildenafil decreased contractility and increased cGMP release. Increased smooth muscle tone and structural changes in the ischemic prostate may suggest a role for prostate ischemia in resistance to urinary flow independent of prostate size.

Topics: Adrenergic alpha-Antagonists; Animals; Arginine; Arteriosclerosis; Blood Flow Velocity; Cyclic GMP; Doxazosin; Electric Stimulation; Iliac Artery; In Vitro Techniques; Ischemia; Laser-Doppler Flowmetry; Male; Muscle Contraction; Muscle, Smooth; Nitric Oxide; Phosphodiesterase Inhibitors; Piperazines; Prostate; Purines; Rabbits; Sildenafil Citrate; Sulfones

Carperitide, a synthetic alpha-human atrial natriuretic peptide (ANP) is a newly developed drug for the treatment of heart failure. However, effects of carperitide on susceptibility to ischemia reperfusion injury are left to be determined. Isolated rat hearts were subjected to Langendorff perfusion. Six hearts received 0.1 microM of carperitide for 10 min, 6 hearts received 1 mM of a NO synthetase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) for 5 min before the infusion of carperitide, 6 hearts received 0.02 microM of a PKC synthetase inhibitor chelerythrine chloride for 5 min before the infusion of carperitide, 6 hearts received 100 microM of a selective mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-dehydroxydecanoate (5HD) before the infusion of carperitide, 6 hearts received 10 microM of a soluble guanylate cyclase inhibitor methylene blue for 5 min before the infusion of carperitide, and 6 hearts served as a control with no drug infusion. All hearts were then subjected to 20 min of global ischemia followed by 120 min of reperfusion. Left ventricular pressures and coronary flow were measured throughout the experiment and infarct size was detected at the end of experiment. Both plasma and tissue cGMP levels were also determined. The results showed: (1) Carperitide significantly reduced infarct size compared to control (26.1 +/- 2.8 vs. 42.7 +/- 2.3%, carperitide vs. control, p < 0.05). This effect was reversed by L-NAME, chelerythrine and 5HD, but not methylene blue. (2) Plasma cGMP levels were increased in carperitide-treated group. This effect was reversed by L-NAME (0.16 +/- 0.03 vs. 1.04 +/- 0.09* vs. 0.28 +/- 0.02 nmol/L, control vs. carperitide vs. L-NAME, *p < 0.01 vs. control). We conclude that preischemic infusion of carperitide exerts cardioprotective effects possibly through NO-PKC dependent pathway followed by mitochondrial KATP channel activation.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Atrial Natriuretic Factor; Benzophenanthridines; Cyclic GMP; Enzyme Inhibitors; Heart; Ischemia; Ischemic Preconditioning, Myocardial; Methylene Blue; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phenanthridines; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Rats; Reperfusion; Time Factors; Tissue Distribution

We examined whether oral folate supplementation would rescue a hypercholesterolemia (HC)-related impairment of ischemia-induced angiogenesis.. Folate protects against endothelial dysfunction, but the effect of folate supplementation on angiogenesis is little known.. Sprague-Dawley rats were divided into four groups. Control rats were fed a normal diet (n = 18); HC rats (n = 18) were fed 2% cholesterol diet; and HC + folate (HC+F) rats were fed an HC diet with oral folate (0.003% in water). The left femoral artery and vein were surgically excised, and angiogenesis in the ischemic limb was evaluated. We also examined the effects of Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, on angiogenesis in the HC+F state.. Laser Doppler blood flow (LDBF) analysis showed lower ischemic/normal LDBF ratio in the HC group than in the control group. Angiographic and histologic analyses on day 14 revealed a smaller angiographic score (p < 0.001) and capillary density (p < 0.001) in the HC group than in controls, which were associated with reduced tissue NOx and cyclic guanosine monophosphate (cGMP) levels. The LDBF ratio, angiographic score, and capillary density were significantly restored in the HC+F group (p < 0.01 vs. HC), which were associated with increased serum folate and tissue NOx and cGMP levels. Finally, L-NAME treatment abolished the beneficial action of folate on angiogenesis in the HC state.. Ischemia-induced angiogenesis was inhibited by HC, which was rescued by oral folate supplementation, at least in part, via an NO-dependent manner.

Topics: Administration, Oral; Animals; Cholesterol; Cholesterol, HDL; Cyclic GMP; Dietary Supplements; Disease Models, Animal; Drug Evaluation, Preclinical; Folic Acid; Hindlimb; Homocysteine; Hypercholesterolemia; Ischemia; Male; Neovascularization, Pathologic; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peripheral Vascular Diseases; Rats; Rats, Sprague-Dawley; Risk Factors; Severity of Illness Index

Endothelium-derived NO has been shown to mediate the mitogenic effect of vascular endothelial growth factor on cultured microvascular endothelium. To evaluate the role of endothelial NO synthase (eNOS) in angiogenesis in the ischemic hindlimb, we engineered an adenovirus containing human eNOS cDNA.. After gene transfer, expression of eNOS in cultured cells was detected by increased intracellular cGMP and nitrate/nitrite levels and NO synthase activity. Adenovirus containing either the eNOS or luciferase gene was injected into the adductor muscle of rat hindlimbs immediately after femoral artery removal. Human eNOS protein was detected throughout the course of the experiment by immunostaining. Significant increases in blood perfusion were monitored by laser Doppler imaging from 2 to 4 weeks after gene delivery in the ischemic hindlimb of rats receiving eNOS compared with control rats receiving the reporter gene. An increase in regional blood flow was also detected after eNOS gene transfer by a fluorescent microsphere assay. eNOS gene delivery in the ischemic hindlimb resulted in significant increases in intracellular cGMP levels and in capillary density identified by anti-CD-31 immunostaining. Angiogenesis was further confirmed in mice after eNOS gene transfer by increased hemoglobin content in Matrigel implants.. Taken together, these results indicate that eNOS enhances angiogenesis and raises the potential of eNOS gene transfer for modulation of vascular insufficiency.

Topics: Adenoviridae; Animals; Capillaries; Cells, Cultured; Cyclic GMP; DNA, Viral; Endothelium, Vascular; Gene Transfer Techniques; Genetic Vectors; Hemoglobins; Hindlimb; Ischemia; Laser-Doppler Flowmetry; Male; Mice; Models, Animal; Neovascularization, Physiologic; Nitric Oxide Synthase; Plasmids; Rats; Regional Blood Flow

A direct role of carbon monoxide (CO), an effector-signaling molecule during heme oxygenase-1 (HO-1) catalysis of heme, in the protection against hepatic ischemia/reperfusion (I/R) injury needs to be established. This study was designed to determine the effects and downstream mechanisms of CO on cold I/R injury in a clinically relevant isolated perfusion rat liver model. After 24 hours of cold storage, rat livers perfused ex vivo for 2 hours with blood supplemented with CO (300 parts per million) showed significantly decreased portal venous resistance and increased bile production, as compared with control livers perfused with blood devoid of CO. These beneficial effects correlated with improved liver function (serum glutamic oxaloacetic transaminase levels) and diminished histological features of hepatocyte injury (Banff's scores). The CO-mediated cytoprotective effects were nitric oxide synthase- and cyclic guanine monophosphate-independent, but p38 mitogen-activated protein kinase (MAPK)-dependent. Moreover, adjunctive use of zinc protoporphyrin, a competitive HO-1 inhibitor, has shown that exogenous CO could fully substitute for endogenous HO-1 in preventing hepatic I/R insult. This study performed in a clinically relevant ex vivo cold ischemia model is the first to provide the evidence that HO-1-mediated cytoprotection against hepatic I/R injury depends on the generation of, and can be substituted by, exogenous CO. The p38 MAPK signaling pathway represents the key downstream mechanism by which CO prevents the I/R insult. In conclusion, regimens that employ exogenous CO should be revisited, as they may have potential applications in preventing/mitigating I/R injury, and thus expanding the liver donor pool for clinical transplantation.

Topics: Animals; Carbon Monoxide; Cold Temperature; Cyclic GMP; Enzyme Activation; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; In Vitro Techniques; Ischemia; Liver; Liver Circulation; Male; Mitogen-Activated Protein Kinases; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury

In a series of canine liver ischemia experiments, we have shown that amelioration of hepatic injury is achievable by the inhibition of vasoconstriction, cytokine production, platelet aggregation, and neutrophil infiltration. Cyclic adenosine diphosphate (cAMP) was considered to be involved in most of these events. In our study, we tested our hypothesis that augmentation of endogenous cAMP by phosphodiesterase (PDE) 3 inhibitor, amrinone (AM), or adenylate cyclase stimulator, NKH477 (NKH), could attenuate ischemia and reperfusion injury of the liver.. Thirty-six beagle dogs were used. They were divided into group CT (untreated control), group AM, group NKH, and group CB (treated by both agents). AM or NKH were administered i.v. 1 hr before ischemia (group preAM and group preNKH) or 15 min before reperfusion (pos-AM and postNKH). Combination group animals were treated only before ischemia. Animal survival, hepatic tissue blood flow, liver enzymes, platelet counts, energy metabolism, hepatic cAMP and cyclic guanosine 3',5'-cyclic monophosphate levels, and histopathology were analyzed.. Two-week animal survival was significantly improved by pre- or posttreatment with either agent. After reperfusion, hepatic tissue blood flow, liver enzyme release, platelet counts, energy metabolism, tissue cAMP levels, and histological architecture were also ameliorated markedly. Combination of both agents induced severe liver damage and lethal hypotension. AM treatment exhibited more protective effects than NKH, particularly when it was given before ischemia. Interestingly, not only cyclic guanosine 3',5'-cyclic monophosphate, were also restored at higher levels after reperfusion by preischemia treatment.. Administration of amrinone or NKH477 maintained hepatic tissue concentrations of cyclic nucleotides, and attenuated ischemia and reperfusion injury of the liver. Thus, regulation of hepatic tissue cyclic nucleotides is an important alternative for prevention of hepatic damage in liver preservation and surgery.

Topics: Adenosine Triphosphate; Amrinone; Animals; Colforsin; Cyclic AMP; Cyclic GMP; Dogs; Energy Metabolism; Female; Hemodynamics; Ischemia; Liver; Liver Circulation; Platelet Count; Portal Vein; Reperfusion Injury

We examined whether endogenous inhibitors of nitric oxide (NO) synthesis are involved in the impaired cavernosal relaxation with ischemia in rabbits. Two weeks after cavernosal ischemia caused by partial vessel occlusion, endothelium-dependent and electrical field stimulation (EFS)-induced neurogenic NO-mediated relaxations, but not sodium nitroprusside (SNP)-induced relaxation, were significantly impaired in the isolated corpus cavernosum. The Ca(2+)-dependent NO synthase (NOS) activity and the basal and stimulated cGMP productions with carbachol or EFS were significantly decreased after ischemia. Supplementation of excess L-arginine partially recovered both of the impaired relaxations. The contents of N(G)-monomethyl-L-arginine (L-NMMA) and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) but not L-arginine and symmetric N(G),N'(G)-dimethyl-L-arginine (SDMA) were increased in the cavernosal tissues after ischemia. Authentic L-NMMA and ADMA but not SDMA concentration dependently inhibited both relaxations without affecting the relaxation produced by SNP in the control. Excess L-arginine abolished the inhibition with L-NMMA and ADMA. These results suggest that the impaired NO-mediated cavernosal relaxations after ischemia are closely related to the decreased NOS activity and the increased accumulation of L-NMMA and ADMA.

Topics: Animals; Arginine; Calcium; Carbachol; Cholinergic Agonists; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Endothelium, Vascular; Enzyme Inhibitors; Iliac Artery; Impotence, Vasculogenic; In Vitro Techniques; Ischemia; Male; Nitric Oxide Synthase; omega-N-Methylarginine; Penis; Rabbits; Vasoconstrictor Agents; Vasodilator Agents

Ischemia is an inciting factor in 50% of incidences of acute renal failure, and it increases the risk of organ rejection after renal transplantation. We have previously demonstrated that resveratrol (RSV) reduces ischemia-reperfusion (I/R) injury of rat kidney both by antioxidant and anti-inflammatory mechanisms. However, a clear morphological demonstration of this activity has not been made. To answer this question we have performed a new set of experiments following the experimental protocol reported below to investigate the effects of I/R injury and RSV pretreatment on kidney morphology by computerized morphometric analysis. Both renal arteries were clamped for 40 minutes in 40 male Wistar rats (b.w. 220 +/- 20 g); 20 rats were pretreated with RSV 1 microM e.v. 40 minutes before clamping. All animals were reperfused for 24 hours and then sacrificed. Histological examination showed tissue conservation in treated rats. I/R-induced glomerular collapse (as revealed by mean glomerular volume and glomerular shape factor) was significantly reduced by RSV pretreatment. Capillary tuft/Bowman's capsule area ratio was enhanced in the I/R group suggesting tubular hypertension. RSV pre-treatments significantly reduced this parameter to the control value. The number of platelet clots in the capillary tuft and tubular necrosis were also reduced by RSV versus I/R group. L-NAME administration worsened both functional and structural damage. Finally, cGMP urinary levels were markedly reduced from 12.1 +/- 8.4 nmol/day to 0.10 +/- 0.10 nmol/day in the I/R group. RSV provided cGMP (5.01 +/- 1.5 nmol/day, P < 0.05). As expected, L-NAME administration significantly reduced cGMP in urine (0.71 +/- 0.6 nmol/day). The present study confirms the protective effect of RSV pretreatment in I/R injury of rat kidney and suggests multiple mechanisms of action.

Topics: Animals; Cyclic GMP; Dose-Response Relationship, Drug; Ischemia; Kidney Diseases; Kidney Glomerulus; Male; Rats; Rats, Wistar; Renal Circulation; Reperfusion Injury; Resveratrol; Stilbenes; Vitis; Wine

Renal ischemia in humans and in experimental animals is associated with a complex and possibly interrelated series of events. In this study, we have investigated the glomerular nitric oxide (NO) production after renal ischemia. Unilateral or bilateral renal ischemia was induced in Wistar rats by clamping one or both renal arteries. NO production was assessed by measuring glomerular production of nitrite, a stable end product of NO catabolism, and NO-dependent glomerular cGMP production and by assessing the glomerular NADPH diaphorase (ND) activity, an enzymatic activity that colocalizes with NO-synthesis activity. Furthermore, we determined the isoform of NO synthase (NOS) implicated in NO synthesis by Western blot and immunohistochemistry. Glomeruli from rats with bilateral ischemia showed elevated glomerular nitrite and cGMP production. Besides, glomeruli from this group of rats showed an increased ND activity, whereas glomeruli from the ischemic and nonischemic rats with unilateral ischemia did not show this increase in nitrite, cGMP, and ND activity. In addition, glomeruli from ischemic kidneys showed an increased expression of endothelial NOS without changes in the inducible isoform. Addition of L-NAME in the drinking water induced a higher increase in the severity of the functional and structural damage in rats with bilateral ischemia than in rats with unilateral ischemia and in sham-operated animals. We can conclude that after renal ischemia, there is an increased glomerular NO synthesis subsequent to an activation of endothelial NOS that plays a protective role in the renal damage induced by ischemia and reperfusion.

Topics: Animals; Blotting, Western; Constriction; Creatinine; Cyclic GMP; Enzyme Inhibitors; Female; Immunohistochemistry; Ischemia; Isoenzymes; Kidney; Kidney Glomerulus; Kidney Tubules; Microscopy, Electron; NADPH Dehydrogenase; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitrites; Rats; Rats, Wistar; Renal Artery

1. We examined the effect of ischaemia on the neurogenic and nitric oxide (NO)-mediated urethral relaxation. 2. Rabbits were divided into control and urethral ischaemia (UI) groups, which was prepared by the partial occlusion of bilateral iliac arteries using blood vessel occluders. 3. Neurogenic and NO-mediated proximal urethral relaxation induced by electrical field stimulation (EFS) was greatly impaired in the UI group, while relaxation by sodium nitroprusside (SNP) as a NO donor showed no difference between the two groups. Pretreatment with L-arginine significantly improved but did not normalize the impaired relaxation in the UI group. Not only basal level, but also stimulated production of cyclic GMP with EFS, were significantly decreased in the UI group. 4. The tissue contents of N(G)-methyl-L-arginine (L-NMA) and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) in the proximal urethra were increased following ischaemia. While L-arginine and symmetric N(G), N'(G)-dimethyl-L-arginine (SDMA) contents remained unchanged. Exogenously applied authentic L-NMA and ADMA (1 -- 100 microM) concentration-dependently inhibited the EFS-induced urethral relaxation in the control group. The inhibition with L-NMA and ADMA was undetectable in the presence of 3 mM L-arginine. 5. The Ca(2+)-dependent NOS activity in the urethra from the UI group was significantly lower than that from the control group and was not restored by an addition of 3 mM L-arginine. 6. These results suggest that the impaired neurogenic and NO-mediated urethral relaxation with ischaemia is closely related to the increased accumulation of L-NMA and ADMA and decreased NOS activity, which would result in an accelerated reduction in NO production/release.

Topics: Animals; Arginine; Blood Pressure; Body Weight; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Iliac Artery; In Vitro Techniques; Ischemia; Male; Muscle Relaxation; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Phenylephrine; Rabbits; Urethra

Recent studies have suggested a proangiogenic effect of angiotensin-converting enzyme (ACE) inhibition. We hypothesized that such a proangiogenic effect of ACE inhibition may be mediated, in part, by bradykinin (BK) B(2)-receptor pathway. This study therefore examined the neovascularization induced by ACE inhibitor treatment in B(2) receptor-deficient mice (B(2)(-/-)) in a model of surgically induced hindlimb ischemia. After artery femoral occlusion, wild-type and B(2)(-/-) mice were treated with or without ACE inhibitor (perindopril, 3 mg/kg/d) for 28 days. Angiogenesis was then quantitated by microangiography, capillary density measurement, and laser Doppler perfusion imaging. The protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) were determined by Western blot. In wild-type animals, vessel density and capillary number in the ischemic leg were raised by 1.8- and 1.4-fold, respectively, in mice treated with ACE inhibitor when compared with the nontreated animals (P<0.01). This corresponded to an improved ischemic/nonischemic leg perfusion ratio by 1.5-fold in ACE inhibitor-treated animals when compared with the untreated ones (0.87+/-0.07 versus 0.59+/-0.05, respectively, P<0.01). Activation of the angiogenic process was also associated with a 1.7-fold increase in tissue eNOS protein level in mice treated with ACE inhibitor (P<0.05 versus control) but not with changes in VEGF protein level. Conversely, ACE inhibition did not affect vessel density, blood flow, and eNOS protein level in ischemic hindlimb of B(2)(-/-) mice. Therefore, proangiogenic effect of ACE inhibition is mediated by B(2)-receptor signaling and was associated with upregulation of eNOS content, independently of VEGF expression.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Blood Flow Velocity; Blood Pressure; Capillaries; Cell Count; Cyclic GMP; Disease Models, Animal; Endothelial Growth Factors; Femoral Artery; Hindlimb; Ischemia; Lymphokines; Male; Mice; Mice, Knockout; Neovascularization, Physiologic; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Receptor, Bradykinin B2; Receptors, Bradykinin; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The activation of the soluble guanylate cyclase and the production of cyclic 3',5'-guanosine monophosphate (cGMP) have been reported as the primary cellular response to nitric oxide (NO) in the nervous system. Previous results indicated, that three-fold sublethal ischemia repeated at 1-h intervals induces damage in gray matter of the spinal cord. However, little is known about the changes of NO/cGMP signal transduction system in gray matter of the spinal cord under conditions of repeated sublethal ischemia.. The aim of this study was to compare the catalytic NOS activity and cGMP concentration in the gray matter regions of lumbosacral spinal cord segments (dorsal horn, zona intermedia, ventral horn) after three (8-, 8-, 9-min) sublethal occlusions repeated at 1-h intervals.. Twenty male rabbits, weighing 2.5-3.5 kg were used in the experiments. They were divided into two experimental groups: (1) control animals (n = 10); (2) animals subjected to three brief (8-, 8-, 9-min) occlusions, each time repeated by reperfusion lasting for 1 h (n = 10). Ischemia of lumbosacral segments was induced by ligation of the abdominal aorta just below the left renal artery (DeGirolami and Zivin, 1982). The catalytic NOS activity was determined by conversion of [3H]-L-arginine to [3H]-L-citrulline according to the method of Bredt and Snyder (1990) slightly modified by Strosznajder and Chalimoniuk (1996). cGMP concentration was assessed by radioimmunoassay method (RIA).. Repeated sublethal ischemia evoked a slight increase in catalytic NOS activity over control values in all gray matter regions. On the other hand, cGMP concentration in gray matter regions has a decreasing character, in a descending order: dorsal horn > zona intermedia > ventral horn. A significant impairment of NO-cGMP signal transduction was detected in the intermediate zone and ventral horns.. Our results indicate that threefold (8-, 8-, 9-min) sublethal ischemia repeated in 1 h intervals of reperfusion causes the impairment of NO/cGMP signal transduction system in gray matter of lumbosacral spinal cord segments and the extent of impairment is region-specific. This finding correlates with the neurological hindlimbs impairment in experimental animals. (Tab. 1, Fig. 2, Ref. 39.)

Topics: Animals; Cyclic GMP; Ischemia; Lumbar Vertebrae; Male; Nitric Oxide; Nitric Oxide Synthase; Rabbits; Reperfusion; Signal Transduction; Spinal Cord

The aim of this study was to test the hypothesis that agents which stabilize the mast cell membrane may modulate the phenotype of the vascular wall in a lung ischemia-reperfusion model, including altering expression of endothelial and leukocyte adhesion receptors and the inducible nitric oxide synthase (NOS-2).. Three sets of rats were given either intravenous saline (group A), ketotifen (group B), or cromolyn (group C), respectively. The left pulmonary artery was ligated temporarily and reopened after 2 hours of ischemia. Then, after a 2-hour period of reperfusion, the left lung was excised. ICAM-1 and NOS-2 were measured at the protein level by Western blotting, and cGMP levels were measured by enzyme-linked immunosorbent assay in the lung tissue specimens for each drug group.. ICAM-1 expressions, determined as the intensity of a given band on the Western blot, were 197+/-59 in group B and 195+/-83 in group C versus 369+/-114 in group A (p = 0.002 for analysis of variance). In contrast with ICAM-1, NOS-2 expression was increased by ketotifen or cromolyn treatment (464+/-82 in group B and 507+/-93 in group C, compared with 377+/-44 for group A, p = 0.007). The finding of increased NOS-2 expression in groups B and C is consistent with the observed increase in tissue cGMP levels in the same groups (1.92+/-0.9 pmol/mL for group A versus 7.8+/-3.5 pmol/mL for group B, and 12.4+/-5.8 pmol/mL for group C, p = 0.0004).. These data establish that mast cell stabilizing agents modulate the vascular phenotype in the setting of pulmonary ischemia and reperfusion by decreasing ICAM-1 expression, augmenting expression of NOS-2, and increasing tissue cGMP levels. As decreasing ICAM-1 expression and increasing cGMP levels have proven useful to limit proinflammatory mechanisms of tissue injury, mast cell stabilizing agents may provide a new therapeutic option to improve organ function in the setting of reperfusion.

Topics: Analysis of Variance; Animals; Anti-Asthmatic Agents; Cell Membrane; Cromolyn Sodium; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Histamine H1 Antagonists; Intercellular Adhesion Molecule-1; Ischemia; Ketotifen; Lung; Male; Mast Cells; Nitric Oxide Synthase; Phenotype; Rats; Rats, Sprague-Dawley; Reperfusion Injury

We have previously shown that PGE(2) enhances recovery of transmucosal resistance (R) in ischemia-injured porcine ileum via a mechanism involving chloride secretion. Because the tyrosine kinase inhibitor genistein amplifies cAMP-induced Cl(-) secretion, we postulated that genistein would augment PGE(2)-induced recovery of R. Porcine ileum subjected to 45 min of ischemia was mounted in Ussing chambers, and R and mucosal-to-serosal fluxes of [(3)H]N-formyl-methionyl-leucyl phenylalanine (FMLP) and [(3)H]mannitol were monitored as indicators of recovery of barrier function. Treatment with genistein (10(-4) M) and PGE(2) (10(-6) M) resulted in synergistic elevations in R and additive reductions in mucosal-to-serosal fluxes of [(3)H]FMLP and [(3)H]mannitol, whereas treatment with genistein alone had no effect. Treatment of injured tissues with genistein and either 8-bromo-cAMP (10(-4) M) or cGMP (10(-4) M) resulted in synergistic increases in R. However, treatment of tissues with genistein and the protein kinase C (PKC) agonist phorbol myristate acetate (10(-5)-10(-6) M) had no effect on R. Genistein augments recovery of R in the presence of cAMP or cGMP but not in the presence of PKC agonists.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Chlorides; Cyclic GMP; Dinoprostone; Drug Synergism; Electric Impedance; Female; Genistein; Ileum; Intestinal Mucosa; Ischemia; Male; Mannitol; N-Formylmethionine Leucyl-Phenylalanine; Swine; Tetradecanoylphorbol Acetate; Tritium

Disruption of endothelial barrier properties with development of noncardiogenic pulmonary edema is a major threat in lung ischemia-reperfusion (I/R) injury that occurs under conditions of lung transplantation. Inhaled nitric oxide (NO) reduced vascular leakage in lung I/R models, but the efficacy of this agent may be limited. We coadministered NO and zaprinast, a cGMP-specific phosphodiesterase inhibitor, to further augment the NO-cGMP axis. Isolated, buffer-perfused rabbit lungs were exposed to 4.5 h of warm ischemia. Reperfusion provoked a transient elevation in pulmonary arterial pressure and a negligible rise in microvascular pressure followed by a massive increase in the capillary filtration coefficient and severe lung edema formation. Inhalation of 10 parts/million of NO or intravascular application of 100 microM zaprinast on reperfusion both reduced pressor response and moderately attenuated vascular leakage. Combined administration of both agents induced no additional vasodilation at constant microvascular pressures, but additively protected against capillary leakage paralleled by a severalfold increase in perfusate cGMP levels. In conclusion, combining low-dose NO inhalation and phosphodiesterase inhibition may be suitable for the maintenance of graft function in lung transplantation by amplifying the beneficial effect of the NO-cGMP axis and avoiding toxic effects of high NO doses.

Topics: Animals; Blood Pressure; Capillary Permeability; Compliance; Cyclic GMP; Female; In Vitro Techniques; Ischemia; Male; Nitric Oxide; Organ Size; Phosphodiesterase Inhibitors; Pulmonary Artery; Pulmonary Circulation; Purinones; Rabbits; Reperfusion Injury

We previously found that increased intravascular pressure decreased ischemic lung injury by a nitric oxide (NO)-dependent mechanism (Becker PM, Buchanan W, and Sylvester JT. J Appl Physiol 84: 803-808, 1998). To determine the role of cyclic nucleotides in this response, we measured the reflection coefficient for albumin (sigma(alb)), fluid flux (), cGMP, and cAMP in ferret lungs subjected to either 45 min ("short"; n = 7) or 180 min ("long") of ventilated ischemia. Long ischemic lungs had "low" (1-2 mmHg, n = 8) or "high" (7-8 mmHg, n = 6) vascular pressure. Other long low lungs were treated with the NO donor (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium -1, 2-diolate (PAPA-NONOate; 5 x 10(-4) M, n = 6) or 8-bromo-cGMP (5 x 10(-4) M, n = 6). Compared with short ischemia, long low ischemia decreased sigma(alb) (0.23 +/- 0.04 vs. 0.73 +/- 0.08; P < 0.05) and increased (1.93 +/- 0.26 vs. 0.58 +/- 0.22 ml. min(-1). 100 g(-1); P < 0.05). High pressure prevented these changes. Lung cGMP decreased by 66% in long compared with short ischemia. Lung cAMP did not change. PAPA-NONOate and 8-bromo-cGMP increased lung cGMP, but only 8-bromo-cGMP decreased permeability. These results suggest that ischemic vascular injury was, in part, mediated by a decrease in cGMP. Increased vascular pressure prevented injury by a cGMP-independent mechanism that could not be mimicked by administration of exogenous NO.

Topics: Animals; Blood Pressure; Capillary Permeability; Cyclic AMP; Cyclic GMP; Hydrazines; Ischemia; Lung; Male; Nitric Oxide; Nitric Oxide Donors; Nucleotides, Cyclic; Respiration, Artificial; Serum Albumin; Water

Endothelium-derived nitric oxide (EDNO) plays an important role in the regulation of angiogenesis, whereas hypercholesterolemia (HC) impairs EDNO release. We examined the hypothesis that HC may inhibit ischemia-induced angiogenesis by inhibition of EDNO in a rat model of unilateral hindlimb ischemia and that oral L-arginine supplementation, a substrate for NO synthase, may prevent HC-related impairment of angiogenesis.. Male Sprague-Dawley rats were fed (A) standard diet (control), (B) 2% high-cholesterol diet (HC group), or (C) high-cholesterol diet with oral L-arginine (2.25% in drinking water) (HC+L-arg group). At 2 weeks of the dietary intervention, unilateral limb ischemia was surgically induced in all animals. Dietary HC groups (B and C) revealed elevated total and LDL cholesterol levels compared with control animals. Laser Doppler blood flow analyses showed significant decreases in the ischemic/normal limb blood flow ratio in the HC group compared with controls (P:<0.05) when followed up until 4 weeks after surgery. Selective angiography and immunohistochemical analyses in the ischemic limb at postoperative day 14 revealed significantly lower angiographic scores (P:<0.01) and capillary densities (P:<0.01) in the HC group than controls, which were associated with decreased tissue contents of NO(x) and cGMP. Oral L-arginine supplementation (HC+L-arg) significantly improved all parameters of the laser Doppler blood perfusion ratio, angiographic scores, and capillary densities (P:<0.01 versus HC group), which were accompanied by significant elevations in serum L-arginine levels and tissue NO(x) and cGMP contents.. Collateral vessel formation and angiogenesis in response to hindlimb ischemia were significantly attenuated in rats with dietary HC. The mechanism may be related to the reduced NO bioactivity in the ischemic tissues. Augmentation of the tissue NO activity by oral L-arginine supplementation restored the impaired angiogenesis in HC.

Topics: Administration, Oral; Animals; Arginine; Body Weight; Cholesterol, Dietary; Collateral Circulation; Cyclic GMP; Endothelium, Vascular; Hindlimb; Hypercholesterolemia; Immunohistochemistry; Ischemia; Laser-Doppler Flowmetry; Lipids; Male; Neovascularization, Physiologic; Nitrates; Nitric Oxide; Nitrites; Rats; Rats, Sprague-Dawley; Regional Blood Flow

Hyperhomocysteinemia (HH) is an independent risk factor for atherosclerosis, including peripheral arterial occlusive disease (PAOD). Because angiogenesis and collateral vessel formation are important self-salvage mechanisms for ischemic PAOD, we examined whether HH modulates angiogenesis in vivo in a rat model of hindlimb ischemia. Rats were divided into 3 groups: the control group was given tap water, the HH group was given water containing L-methionine (1 g x kg(-1) x d(-1)), and the HH+L-arg group was given water containing methionine (1 g x kg(-1) x d(-1)) and l-arginine (2.25 vol%). At day 14 of the dietary modifications, the left femoral artery and vein were excised, and the extent of angiogenesis and collateral vessels in the ischemic limb were examined for 4 weeks. Plasma homocysteine levels significantly increased (P:<0.001), and plasma and tissue contents of nitrite+nitrate as well as tissue cGMP levels significantly decreased in the HH group compared with the control group (P:<0.01). Laser Doppler blood flowmetry (LDBF) revealed a significant decrease in the ischemic/normal limb LDBF ratio in the HH group at days 7, 14, 21, and 28 (P:<0.01 versus control). Angiography revealed a significant decrease in the angiographic score in the HH group at day 14 (P:<0.001 versus control). Immunohistochemistry of ischemic tissue sections showed a significant reduction in the capillary density in the HH group (P:<0. 001 versus control). Oral l-arginine supplementation in rats with HH (HH+L-arg) restored the decreased plasma and tissue nitrite+nitrate and cGMP contents (P:<0.05) as well as angiogenesis, as assessed by LDBF (P:<0.05 versus HH), angiographic score (P:<0.01 versus HH), and capillary density (P:<0.001 versus HH). In summary, HH impaired ischemia-induced angiogenesis and collateral vessel formation in a rat model of hindlimb ischemia in vivo. The mechanism of the HH-induced impairment of angiogenesis might be mediated in part by a reduced bioactivity of endogenous NO in the HH state.

Topics: Angiography; Animals; Arginine; Blood Pressure; Body Weight; Collateral Circulation; Cyclic GMP; Disease Models, Animal; Heart Rate; Hindlimb; Homocysteine; Hyperhomocysteinemia; Immunohistochemistry; Ischemia; Laser-Doppler Flowmetry; Muscle, Skeletal; Neovascularization, Physiologic; Nitrates; Nitrites; Rats; Regional Blood Flow; Time Factors

It has been reported that nitric oxide raises c-GMP in the vascular muscle to cause vasodilation and to improve blood flow in the retina. Consequently, a diverse group of potential nitric oxide (NO) donors were synthesized and evaluated for their effectiveness in improving the retinal function after ischemic insult. These compounds include an NO carrier, N-acetyl-S-nitrosoglutathione (RVC-593), several NO donors such as N-nitropyrazole derivatives (RVC-595, RVC-596, RVC-597, RVC-598, and RVC-599) and two fused N-heterocycles, 4H-[1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione 1-oxides, (RVC-600 and RVC-601). Most of the compounds demonstrated the pharmacological activity in the ophthalmic model, except the pyrazole derivatives (RVC-595, RVC-596 and RVC-599) that bear bulky aromatic substituents at the R2-position.

Topics: Animals; Ciliary Arteries; Cyclic GMP; Dark Adaptation; Electroretinography; Female; Injections, Intraperitoneal; Ischemia; Nitric Oxide; Nitric Oxide Donors; Photic Stimulation; Rats; Rats, Long-Evans; Recovery of Function; Retina; Retinal Vessels; Vasodilation

Ventilation during ischemia attenuates ischemia-reperfusion lung injury, but the mechanism is unknown. Increasing tissue cyclic nucleotide levels has been shown to attenuate lung ischemia-reperfusion injury. We hypothesized that ventilation prevented increased pulmonary vascular permeability during ischemia by increasing lung cyclic nucleotide concentrations. To test this hypothesis, we measured vascular permeability and cGMP and cAMP concentrations in ischemic (75 min) sheep lungs that were ventilated (12 ml/kg tidal volume) or statically inflated with the same positive end-expiratory pressure (5 Torr). The reflection coefficient for albumin (sigmaalb) was 0.54 +/- 0.07 and 0.74 +/- 0. 02 (SE) in nonventilated and ventilated lungs, respectively (n = 5, P < 0.05). Filtration coefficients and capillary blood gas tensions were not different. The effect of ventilation was not mediated by cyclic compression of alveolar capillaries, because negative-pressure ventilation (n = 4) also was protective (sigmaalb = 0.78 +/- 0.09). The final cGMP concentration was less in nonventilated than in ventilated lungs (0.02 +/- 0.02 and 0.49 +/- 0. 18 nmol/g blood-free dry wt, respectively, n = 5, P < 0.05). cAMP concentrations were not different between groups or over time. Sodium nitroprusside increased cGMP (1.97 +/- 0.35 nmol/g blood-free dry wt) and sigmaalb (0.81 +/- 0.09) in nonventilated lungs (n = 5, P < 0.05). Isoproterenol increased cAMP in nonventilated lungs (n = 4, P < 0.05) but had no effect on sigmaalb. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester had no effect on lung cGMP (n = 9) or sigmaalb (n = 16) in ventilated lungs but did increase pulmonary vascular resistance threefold (P < 0.05) in perfused sheep lungs (n = 3). These results suggest that ventilation during ischemia prevented an increase in pulmonary vascular protein permeability, possibly through maintenance of lung cGMP by a nitric oxide-independent mechanism.

Topics: Animals; Capillary Permeability; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; In Vitro Techniques; Ischemia; Lung; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroprusside; Pulmonary Circulation; Respiration, Artificial; Sheep; Vascular Resistance; Vasodilator Agents

Atherosclerotic vascular lesions are considered to be a major cause of ischemic diseases, including myocardial infarction and stroke. Platelet adhesion and aggregation during ischemia-reperfusion are thought to be the initial steps leading to remodeling and reocclusion of the postischemic vasculature. Nitric oxide (NO) inhibits platelet aggregation and smooth muscle proliferation. A major downstream target of NO is cyclic guanosine 3', 5'-monophosphate kinase I (cGKI). To test the intravascular significance of the NO/cGKI signaling pathway in vivo, we have studied platelet-endothelial cell and platelet-platelet interactions during ischemia/reperfusion using cGKI-deficient (cGKI-/-) mice. Platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation.

Topics: Animals; Blood Platelets; Cell Adhesion; Cell Adhesion Molecules; Cell Size; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelium, Vascular; In Vitro Techniques; Ischemia; Mice; Mice, Knockout; Microcirculation; Microfilament Proteins; Nitric Oxide; Phosphoproteins; Phosphorylation; Platelet Aggregation; Platelet Aggregation Inhibitors; Serotonin

Recent studies have reported that nitric oxide (NO) acts as a cytoprotective mediator in ischemia-reperfusion (IR) lung injury. We hypothesized that the addition of L-arginine to the perfusate would attenuate the increases in microvascular permeability and pulmonary vascular resistance.. Isolated rabbit lungs were reperfused for 60 min after 120 min warm ischemia. Lung injury was assessed using the fluid filtration coefficient (Kf), pulmonary vasucular resistance (PVR) before ischemia and after reperfusion, and a wet-to-dry lung weight ratio (W/D).. The Kf of the control group (without L-arginine) was significantly increased after reperfusion. Lungs perfused with L-arginine showed attenuation of the IR-induced increases in Kf and PVR. Addition of Nomega-nitro-L-arginine (L-NA), a NO synthase inhibitor, to the perfusate reduced the beneficial effects of L-arginine. The lungs perfused with dibutyryl-cyclic GMP (dbcGMP) showed attenuation of IR-induced increases in Kf and PVR. There were no significant differences in the W/D ratio between these groups.. These results demonstrate that L-arginine has beneficial effects on IR lung injury, perhaps due to enhancement of endothelial cGMP levels.

Topics: Animals; Arginine; Body Fluids; Capillary Permeability; Cyclic GMP; Enzyme Inhibitors; Hemodynamics; In Vitro Techniques; Ischemia; Lung; Nitroarginine; Pressure; Pulmonary Circulation; Rabbits; Reperfusion Injury; Trachea; Vascular Resistance

A chronic partially ischemic state may alter the skeletal muscle response to acute ischemia and free radical formation.. In order to investigate this hypothesis, a chronic ischemic state was established by ligating the right femoral artery of four mongrel dogs. ABIs were decreased from 1.05 +/- 0.25 preligation to 0.54 +/- 0.14 at 6 weeks (P = 0.04). At the end of 8 weeks, the hindlimb was subjected to 3 h of acute ischemia by clamping the iliac artery. The clamp was then released for 2 h of reperfusion. Plasma samples from the right iliac vein were taken during the ischemia-reperfusion period for analysis of cGMP. Tibialis anterior biopsies for Western analysis of eNOS and iNOS were taken upon completion of reperfusion. Comparisons to control dogs subjected to the acute ischemia and reperfusion without prior femoral artery ligation were made.. cGMP levels were increased in the controls at 3 h of ischemia (3539 +/- 350) and 2 h of reperfusion (2880 +/- 269). The chronic ischemia group did not develop a corresponding increase in cGMP at 3 h of ischemia (2762 +/- 251) or after 2 h of reperfusion (2102 +/- 130). Western analysis of eNOS and iNOS revealed similar levels in both groups. Analysis of eNOS revealed 0.6429 +/- 0.086 and 0.5916 +/- 0.072 (densitometric units +/- SEM) for study and control dogs, respectively. Analysis of iNOS revealed 0.3401 +/- 0.067 and 0.2475 +/- 0.066 for study and control dogs, respectively.. Previous ligation of the femoral artery resulting in chronic partial ischemia in this model demonstrated no increase in cGMP following acute ischemia that was not accompanied by a change in eNOS or iNOS levels. Nitric oxide activity is reflected by cGMP levels, which may increase in response to free radicals in the acute setting of complete ischemia.

Topics: Acute Disease; Animals; Blotting, Western; Chronic Disease; Cyclic GMP; Densitometry; Dogs; Hindlimb; Ischemia; Muscle, Skeletal; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Reference Values; Time Factors

FK409 is the first spontaneous nitric oxide (NO) donor known to increase plasma cyclic guanosine 3',5'monophosphate levels. In this study, we evaluated the effect of FK409 on pulmonary ischemia-reperfusion injury in an in situ warm ischemia canine model. Fourteen dogs were divided into two groups, and the FK409-treated group was given 5 micrograms/kg per min FK409. Warm ischemia was induced for 3 h. The arterial partial pressure of oxygen (PaO2), arterial oxygen saturation (SaO2), cardiac output (CO), left pulmonary vascular resistance (L-PVR), and endothelin-I (ET-I) were measured. A histologic study was performed, and polymorphonuclear neutrophils (PMNs) were also counted. The PaO2, SaO2, and L-PVR levels and PMNs after 30 min of reperfusion, ET-I after 2 h of reperfusion, and the 7-day survival rate were significantly (P < 0.05) better in the FK409-treated group than in the control group. The histologic damage was reduced in the FK409-treated group compared to the control group. FK409 appears to have a protective effect in ischemia-reperfusion injury of the lung.

Topics: Animals; Cardiac Output; Cyclic GMP; Dogs; Drug Evaluation, Preclinical; Endothelin-1; Ischemia; Lung; Nitric Oxide Donors; Nitro Compounds; Oxygen; Partial Pressure; Pulmonary Edema; Reperfusion Injury

We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction.

Topics: Animals; Arginine; Cyclic GMP; Endothelial Growth Factors; Hemodynamics; Iliac Artery; Ischemia; Lymphokines; Male; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Nitric Oxide Synthase; Rabbits; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The aim of our studies was to investigate hormonal prevention of hepatic preservation damage by the atrial natriuretic peptide (ANP) and the mechanisms involved. Isolated perfusion of rat livers was performed in a nonrecirculating fashion. Twenty minutes of preischemic perfusion was performed with or without different concentrations of ANP, followed by 24-hour storage in cold University of Wisconsin (UW) solution. Two hundred nanomoles of ANP prevented hepatocellular damage during a 2-hour reperfusion period as indicated by a marked attenuation of the sinusoidal efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), and by reduced Trypan blue uptake. Furthermore, postischemic bile flow as an indicator of liver function was significantly improved by about 60% with 200 nmol/L ANP. No protection was conveyed by 20 nmol/L ANP nor by pretreatment with 200 nmol/L ANP for only 10 minutes. The effects of ANP seemed to be mediated by the guanylate cyclase-coupled A (GC-A) receptor and cyclic guanosine monophosphate (cGMP): whereas expression of both GC-A and GC-B receptors as well as of the GC-C receptor was found, cGMP did protect from ischemia-reperfusion damage, but selective ligands of the B and C receptor did not. To begin to determine the mechanisms of ANP-mediated protection, different parameters were investigated: ANP had no effect on portal pressure as an indicator of hepatic circulation, nor on intracellular energy depletion determined by adenosine nucleotide concentration. However, the marked augmentation of nuclear factor kappaB (NF-kappaB) binding activity during reperfusion was prevented in ANP-pretreated livers. In conclusion, pretreatment with ANP protects the rat liver from cold ischemia-reperfusion damage. This effect is mediated via the GC-A receptor and cGMP, and may be linked to an influence of ANP on NF-kappaB activation. Thus, ANP signaling via the GC-A receptor should be considered as a new pharmacological target to prevent preservation injury of the liver.

Topics: Animals; Atrial Natriuretic Factor; Cold Temperature; Cyclic GMP; Guanylate Cyclase; Ischemia; L-Lactate Dehydrogenase; Liver; Male; NF-kappa B; Purine-Nucleoside Phosphorylase; Rats; Rats, Sprague-Dawley; Receptors, Atrial Natriuretic Factor; Reperfusion Injury

Local activation of polymorphonuclear leukocytes (PMNLs) is considered an important aspect of the pathogenesis of intermittent claudication, although concrete mechanisms of their effects on circulatory homeostasis in peripheral atherosclerotic disease remain unclear. This study evaluated the ability of PMNLs to deactivate nitric oxide (NO), a key regulator of regional circulation, as a possible factor determining PMNL involvement into ischemic disorders in patients who have intermittent claudication before and after vascular reconstruction.. A total of 57 patients who had peripheral occlusive disease in an aortofemoral segment before surgical treatment (group 1) and 65 patients who had similar occlusive lesions and other clinical and demographic data 6 to 12 months after undergoing inflow vascular reconstruction (group 2) were examined. All patients from group 2 had anatomically patent grafts; their satisfaction and level of function after surgical treatment were assessed by a five-point questionnaire. The sex- and age-matched control group included 35 subjects. NO activity was bioassayed by measuring its ability to increase cyclic guanosine monophosphate (cGMP) accumulation in rat fetal lung-cultured fibroblasts (RFL-6 cells). The ability of PMNLs to deactivate NO was characterized as the percent decrease in NO-induced cGMP accumulation in RFL-6 cells.. Stimulated PMNLs caused inhibition of the activity of authentic NO; accumulation of cGMP induced by sodium nitroprusside was not affected. PMNLs from patients with peripheral atherosclerotic disease either before or after vascular reconstruction had a more marked capacity of NO inactivating than the cells from healthy subjects. For both groups of patients, levels of PMNL-induced NO deactivation were higher for patients with diabetes, and especially both diabetes and arterial hypertension. For both groups of patients, there was no correlation between levels of PMNL-induced NO deactivation and resting ankle-brachial indexes (ABIs). In contrast, close correlation was revealed between levels of PMNL-induced NO deactivation and postexercise ABIs and percent decrease in resting ABIs after exercise in patients evaluated either before or after surgical treatment.. The ability of stimulated PMNLs to deactivate NO is elevated in peripheral occlusive disease and may be implicated in the pathogenesis of intermittent claudication. In patients who underwent successful recanalization of magistral arteries, levels of PMNL-induced NO deactivation remained higher than in control subjects. The increase in the ability of PMNL to deactivate NO positively correlated to ABI decreases after exercise in patients with peripheral occlusive disease either before or after surgical treatment.

Topics: Animals; Aortic Diseases; Arteriosclerosis; Blood Circulation; Blood Pressure; Case-Control Studies; Cells, Cultured; Cyclic GMP; Diabetes Mellitus; Female; Femoral Artery; Fibroblasts; Homeostasis; Humans; Hypertension; Intermittent Claudication; Ischemia; Lung; Male; Middle Aged; Neutrophil Activation; Neutrophils; Nitric Oxide; Nitroprusside; Patient Satisfaction; Peripheral Vascular Diseases; Rats; Vascular Patency

Nitric oxide (NO), identified as the mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory conditions, especially in ischemia/reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion-influenced serum NO and c-GMP concentrations in the flap. In addition, we also investigated the premedicated effects of the NO synthase inhibitor and heparin on serum NO and c-GMP concentrations in skin flap ischemia/reperfusion. Serum NO concentration after 15, 30, 45, and 60 minutes of ischemia followed by reperfusion significantly increased compared with that in nonischemic control and elevated flaps. On the contrary, serum NO concentration was suppressed in L-NAME or aminoguanidine pretreated animals with ischemic group. Administration of heparin increased the serum NO concentration in elevated flaps, but suppressed it in ischemic flaps followed by reperfusion. The changes in serum c-GMP and NO concentrations were related in all of the experimental groups. These results suggest that NO may be derived from vascular endothelial cells and dilate peripheral vessels in compensation for ischemia.

Topics: Animals; Cyclic GMP; Endothelium, Vascular; Epigastric Arteries; Guanidines; Heparin; Ischemia; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Rabbits; Reperfusion; Surgical Flaps

For the first time, the authors report an intimate link between kidney and heart functions as it pertains to the regulation of stress protein gene expression in the heart. They show that the stress on the target organ, the kidney, is translated into a response in the cardiovascular system, as reflected by the induction of heme oxygenase (HO)-1 gene expression, which, in turn, may be a cellular defense response as suggested by an increase in cGMP level in the heart, and an increase in the rate of bilirubin formation by the kidney and the heart. HO-1 is a stress protein (HSP32) and, together with HO-2, catalyzes oxidation of the heme molecule to generate CO, a likely signal molecule for the generation of cGMP, and bilirubin, an antioxidant. Specifically, bilateral renal ischemia for 30 min caused a 3-fold increase in the approximately 1.8-kb HO-1 mRNA in the heart within 4 h after reperfusion and remained essentially at this level for 24 h, at which point, a 2.6-fold increase in HO-1 mRNA in the descending aorta was also detected. Heart HO-1 mRNA remained elevated for more than 48 h; in contrast, at the 48-h time point, the transcript level in the kidney, which had increased by 10-fold 24 h after reperfusion, had returned to the control level. Neither in the heart nor in the kidney did HO-2 transcripts (approximately 1.3 and 1.9 kb) respond to renal ischemia/ reperfusion. The increase in heart HO-1 transcript level was accompanied by an increase in HO-1 protein, as judged by Western blot and immunohistochemical analysis, and in enzyme activity, as judged by bilirubin formation. In addition, cGMP concentration in the heart was elevated when measured at 24 h and 48 h after reperfusion of the kidney, in the absence of an increase in the activity of NO. Data suggest that hemodynamic stress caused by the occlusion of the renal artery is responsible for activation of HO-1 gene expression in the heart. An argument is made for the role of HO-1 in the defense mechanisms of the heart pertaining to the enzyme's function in a hemoprotein regulatory capacity, along with the biological activity of its products.

Topics: Animals; Cyclic GMP; Gene Expression; Heme Oxygenase (Decyclizing); Immunohistochemistry; Ischemia; Kidney; Myocardium; Rats; Up-Regulation

Nitric oxide (NO), identified as a mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory diseases, especially ischemia-reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion influences serum NO concentrations. In addition, the author investigated the effects of NO synthase inhibitors and heparin on skin flap ischemia. Serum NO concentrations after 15, 30, 45, and 60 minutes of ischemia followed by reperfusion were significantly increased compared with non-ischemic controls and elevated flaps. On the other hand, serum NO concentrations were suppressed in nitro-amino-methyl-L-arginine- and aminoguanidine-treated animals. Furthermore, administration of heparin increased serum NO concentrations in controls and animals with elevated flaps, but decreased serum NO concentrations in ischemic flaps with subsequent reperfusion. These results suggest that NO is one of the factors responsible for ischemia-reperfusion injury and that NO synthase inhibitors and heparin may protect against such injury.

Topics: Animals; Cyclic GMP; Enzyme Inhibitors; Guanidines; Heparin; Ischemia; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Rabbits; Reperfusion Injury; Skin; Surgical Flaps

To gain further insights into the greater susceptibility to acute ischemic renal injury (ARF, 45 minutes of renal arteries clamping) of old rats (O, 24-26 months) as against young ones (Y, 3-4 months), and the possible role of endothelium derived relaxing factor (EDRF) and its influence on ischemic renal function.. Basal renal dynamics was compared between O and Y.. Two hours after ischemia, the decrease of GFR RPF, and UV was more severe in O than in Y. Urine cGMP was significantly dropped in both groups. Intravenous administration of L-arginine increasld urine cGMP of the O as well as the Y, with that of the Y higher than that in O. GFR, RPF, UV were elevated significantly in the O. Simultaneous administration of L-NNA, the analogue of L-Arginine could partially antagonize its effect on elevating RPF. RT-PCR technique was used to detect renal inducible nitric oxide synthase (iNOS) expression. Although the ischemic kidney was capable of expressing iNOS mRNA in the presence of L-arginine after ischemia, the production of NO in the old may not be regulated at the transcriptional level, other factors such as NOS enzyme activity, availability of L-arginine and O2, metabolism of NO after its production were suspected to be involved.

Topics: Aging; Animals; Arginine; Cyclic GMP; Ischemia; Kidney; Male; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Wistar; Renal Agents; Reperfusion Injury

The hypothesis was tested that plasma from ischemic hindlimbs facilitates hypertension. Ischemia-induced hypertension was generated in rats by infrarenal aortic cross clamping for 5 h after which plasma was obtained from femoral vein blood. In vitro contractile activity of naive aortic rings incubated for 2 h in plasma collected from ischemic rats demonstrated reduced relaxation to acetylcholine and nitroglycerin. Methylene blue (10(-5) M) induced greater contraction in rings incubated in control vs. ischemic plasma, suggesting that endogenous guanylate cyclase activity is decreased by ischemic plasma. However, 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP) relaxed equally strips incubated in ischemic or control plasma. Acetylcholine-induced nitrite release was significantly lower in ischemic vs. control plasma-incubated strips (8.6 +/- 2.7 vs. 28.2 +/- 2.3 ng/10 mg tissue wt, respectively). The impaired relaxation to acetylcholine in ischemic plasma-incubated rings was significantly increased by L-arginine but not by prior treatment of ischemic plasma with heating or superoxide dismutase and catalase. These findings suggest the impaired relaxation is mediated through inhibition of the nitric oxide-cGMP pathway. Prolonged blunting of vasodilation by ischemic plasma may therefore contribute to maintenance of a sustained vasoconstriction and ischemic hypertension.

Topics: Acetylcholine; Animals; Aorta; Blood Physiological Phenomena; Blood Vessels; Cyclic GMP; Hindlimb; In Vitro Techniques; Ischemia; Methylene Blue; Nitric Oxide; Nitroglycerin; Rats; Rats, Sprague-Dawley; Reference Values

Topics: Analysis of Variance; Animals; Arginine; Cyclic GMP; Ischemia; Kidney; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

To elucidate the vascular endothelial function at pulmonary ischemia and reperfusion, cyclic GMP (cGMP) release in lung perfusate at reperfusion after ischemia was examined as a marker of nitric oxide (NO) release from the endothelium in an in situ perfused rabbit lung. The lung was perfused from the pulmonary artery with Krebs-Henselite buffer and cGMP in the lung effluent from the left ventricle was assayed in a time-dependent manner. The inhibition of cGMP release by NG-nitro-L-arginine methyl ester and the reversal of the effect by L-arginine indicated that cGMP released into the perfusate was elicited by NO from the endothelium. There was a significant decrease in cGMP release during reperfusion after warm ischemia (30 min and 60 min) compared with the immediately perfusion without intervening ischemia. These data suggest that NO release is impaired by the endothelial dysfunction at reperfusion after warm ischemia in the perfused rabbit lung and the biochemical studies of the perfusate is useful in assessing the vascular endothelial function.

Topics: Animals; Arginine; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Ischemia; Lung; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rabbits; Reperfusion

Platelets have been implicated in the pathophysiology of ischemia-reperfusion injury. In this study, antiplatelet effects of cyclic GMP (cGMP)- and cyclic AMP (cAMP)-mediated agents were evaluated in renal ischemia in pentobarbital-anesthetized rats. Renal ischemia was induced by unilateral occlusion of the left renal artery (40 min) followed by reperfusion (30 min) with the contralateral kidney serving as control. 111Indium-labeled platelets, drugs or vehicle were administered 30 min before induction of renal ischemia. Occlusion of the left renal artery for 20, 40 or 60 min resulted in a 100, 300 and 600% increase (over contralateral right kidney) in the platelet-associated 111indium activity in the ischemic kidney. In all subsequent studies the kidney was occluded for 40 min to test the antiplatelet activity of individual agents. 8-Br-cGMP (0.1 and 0.3 mg/kg/min i.v.), zaprinast (0.1 mg/kg/min i.v.) and sodium nitroprusside (0.003 and 0.01 mg/kg/min i.v.) significantly attenuated platelet accumulation in renal ischemia, whereas 8-Br-cAMP (0.3 mg/kg/min i.v.) or milrinone (0.1 mg/kg i.v. bolus, plus 0.01 mg/kg/min) did not. Minoxidil (0.01 and 0.03 mg/kg/min i.v.), a vasodilator which produced equihypotensive effects as the cGMP-mediated agents, and milrinone failed to prevent platelet accumulation. These results demonstrate that modulation of the platelet function by cGMP agents can be dissociated from their blood pressure lowering effects. cGMP is known to inhibit both platelet adhesion and aggregation, whereas cAMP is only active against aggregation. The present findings provide further evidence that cGMP-mediated drugs may afford effective antiplatelet action in an in vivo model of ischemia-reperfusion injury.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Blood Platelets; Cyclic AMP; Cyclic GMP; Ischemia; Kidney; Male; Nitroprusside; Platelet Aggregation Inhibitors; Purinones; Rats; Reperfusion

Topics: Adult; Aged; Animals; Arteriosclerosis Obliterans; Cyclic AMP; Cyclic GMP; Humans; Ischemia; Leg; Male; Middle Aged; Muscles; Nucleotides, Cyclic; Rats; Spectrometry, Gamma; Thromboangiitis Obliterans

Pulmonary vascular resistance is significantly increased in the transplanted lung. We hypothesized that the ischemic or reperfusion injuries incurred by the transplanted lung may produce pulmonary vasomotor dysfunction, which in turn may produce increased pulmonary vascular resistance. In a dog model of autologous lung transplantation, the purpose of this study was to examine the following mechanisms of pulmonary vasomotor control and to relate each of them to cold ischemia and to reperfusion: (1) endothelium-dependent cyclic guanosine monophosphate-mediated vasorelaxation (response to acetylcholine 10(-6) mol/L), (2) endothelium-independent cyclic guanosine monophosphate-mediated vasorelaxation (response to sodium nitroprusside 10(-6) mol/L), and beta-adrenergic cyclic adenosine monophosphate-mediated vasorelaxation (response to isoproterenol 10(-6) mol/L). Autologous right lung transplantation was performed in five dogs. At each of three times, two third-order pulmonary arteries were dissected from each transplanted lung and studied: control (immediately after harvest), cold ischemia (3 hours in 4 degrees C saline solution), and cold ischemia plus reperfusion (1 hour after lung reimplantation). The vasorelaxing effects of acetylcholine, sodium nitroprusside, and isoproterenol were studied in isolated pulmonary arterial rings, suspended on fine wire tensiometers in individual organ chambers. Statistical analysis was by analysis of variance. Results demonstrated significant dysfunction of beta-adrenergic cyclic adenosine monophosphate-mediated relaxation after cold ischemia alone, and this dysfunction was exacerbated by reperfusion. Endothelium-dependent cyclic guanosine monophosphate-mediated relaxation was not impaired by cold ischemia alone but was significantly impaired by reperfusion. Endothelium-independent cyclic guanosine monophosphate-mediated relaxation was not impaired by cold ischemia or reperfusion. We conclude that cold ischemia and reperfusion each produce different patterns of pulmonary vasomotor dysfunction. Cumulatively, such dysfunction may contribute to increased pulmonary vascular resistance in the transplanted lung.

Topics: Animals; Cyclic AMP; Cyclic GMP; Dogs; Ischemia; Lung; Lung Transplantation; Muscle, Smooth, Vascular; Reperfusion Injury; Vascular Resistance; Vasomotor System

Recent data have demonstrated that inhibition of nitric oxide synthesis exacerbated the mucosal injury associated with reperfusion of the postischemic intestine. In this study, using a feline 1-h intestinal ischemia followed by reperfusion model, we tested the possibility that exogenous sources of nitric oxide may prevent the reperfusion-induced mucosal barrier disruption and examined the mechanisms involved. Mucosal barrier integrity was assessed by determining 51Cr-EDTA clearance from blood to lumen. Intestinal blood flow and resistance were also determined. Reperfusion after 1 h of ischemia significantly increased 51Cr-EDTA clearance (0.05 +/- 0.01 to 0.35 +/- 0.07 ml.min-1.100 g-1) and decreased intestinal blood flow by 50%. Exogenous sources of nitric oxide including SIN-1, CAS-754, and nitroprusside as well as exogenous L-arginine all reduced reperfusion-induced mucosal barrier dysfunction without improving intestinal blood flow. Inhibition of endogenous nitric oxide with NG-nitro-L-arginine methyl ester between 1 and 2 h of reperfusion further augmented the rise in mucosal permeability associated with ischemia-reperfusion. Addition of the permeable analogue of guanosine 3',5'-cyclic monophosphate, 8-bromoguanosine 3',5'-cyclic monophosphate, improved reperfusion-induced intestinal blood flow significantly but did not provide protection against mucosal barrier disruption associated with the first hour of ischemia-reperfusion. Exogenous sources of nitric oxide can reduce reperfusion-induced mucosal barrier dysfunction independent of alterations in intestinal blood flow.

Topics: Animals; Cats; Cyclic GMP; Intestinal Mucosa; Intestines; Ischemia; Molsidomine; Nitric Oxide; Nitroprusside; Permeability; Regional Blood Flow; Reperfusion Injury; Sydnones

Follow-up clinical results have been analyzed for 564 patients with occlusions of visceral arteries, which presented clinically as symptoms of chronic pancreatitis in 115 patients. As a rule, chronic pancreatic ischemia leads to exocrine hyposecretion evident from the derangement of cyclonucleotide metabolism. There were also metabolic shifts in pancreatic cells related to hypoxia. Intravital biopsies revealed dystrophy of the acinar tissue as well as inter- and intralobular infiltrates of the connective and fatty tissue.

Topics: Adult; Amylases; Cyclic AMP; Cyclic GMP; Humans; Ischemia; Lipase; Middle Aged; Pancreas; Trypsin; Trypsin Inhibitors

The protective effect of prostaglandin I2 (PGI2) on ischemia-induced liver cell injury was investigated during 60-min, 75-min, and 90-min liver ischemia. Vehicle-treated rats tolerated the 75-min hepatic ischemia poorly. Only 25% of the rats in this group survived more than 7 days. However, the survival rate of PGI2-treated rats (350 ng/kg/min) significantly improved to 67%. Liver cell organelles were well-preserved by the PGI2 treatment. Adenosine triphosphate levels in the liver of the PGI2-treated rats were significantly higher than those of vehicle-treated rats at 60 min of reoxygenation following 75-min ischemia. Cyclic 3'-5' adenosine monophosphate levels markedly increased during 60-min PGI2 infusion. Cyclic 3'-5' guanosine monophosphate levels also significantly increased during the PGI2 infusion and were still higher than those of vehicle-treated rats at the end of the 75-min ischemia. Although the exact cytoprotective mechanism of PGI2 at the cellular level is still unclear, our results demonstrate that elevated ATP and cyclic nucleotides levels play an important role in liver cell preservation during ischemia.

Topics: Adenosine Triphosphate; Animals; Cyclic AMP; Cyclic GMP; Epoprostenol; Glycine; Ischemia; Liver; Male; Prostaglandins; Rats; Rats, Inbred Strains

Topics: Adaptation, Ocular; Animals; Cyclic AMP; Cyclic GMP; Dark Adaptation; Eye; Ischemia; Sciuridae; Time Factors

Hepatic glycogen metabolism in aerobic and hypoxic conditions has been assessed with respect to glycogenolysis, phosphorylase alpha activity and nucleotide content. Insulin did not inhibit glycogen breakdown nor stimulate lipogenesis in the aerobic perfused liver. Partial ischaemia induced glycogen breakdown, release of glucose and changes in nucleotide content in the perfused liver. Phosphorylase alpha content increased within 2 min in response to total ischaemia, in vivo and in the perfused liver. This change was paralleled by an increase in hepatic AMP. Glycogen synthase alpha activity decreased, as did the hepatic content of both cyclic AMP and cyclic GMP.

Topics: Adenine Nucleotides; Aerobiosis; Animals; Carbohydrate Metabolism; Cyclic AMP; Cyclic GMP; Glycogen Synthase; Hypoxia; In Vitro Techniques; Ischemia; Liver Glycogen; Male; Phosphorylase a; Rats

A great deal of knowledge has been gained concerning the activation of adenylate and guanylate cyclase in epidermal cells. Adenylate cyclase is activated by 4 different independent receptors-responding respectively to catecholamine (beta), to prostaglandins (E), to histamine (H2), and to adenosine and it phosphorylated derivatives. Upon activation, each of these receptors becomes unresponsive to further stimulation by its specific stimulator. Guanylate cyclase, on the other hand, is activated by histamine (H1) and epidermal growth factor (EGF). Unlike EGF, the histamine activation is extremely rapid (less than 5 minutes). Epidermal cells are permeable (leak) to cyclic GMP but not cyclic AMP. When the skin is traumatized or injured in any way (even by intradermal injection) there is a sudden catastrophic change in the intracellular levels of the cyclic nucleotides (and of ATP). Cyclic AMP rapidly rises to perhaps 5-10 times its normal resting level while cyclic GMP falls to 10-20% of its level in vivo. The rise in cyclic AMP is due to activation of adenylate cyclase while the fall in cyclic GMP is due in major part to activation of cyclic GMP phosphodiesterase (and perhaps the fall in ATP is due to activation of ATPase). The changes in ATP and cyclic AMP can be reversed by incubating the tissue in a buffered salt solution containing glucose, but this does not normalize the cyclic GMP content. The fall in cyclic GMP can be prevented by a phosphodiesterase inhibitor (IBMX ). This series of events has been called the "ischemia effect." However, it implies that a lack of oxygen is at fault, and that has not been shown to be the case. Its underlying cause and possible physiologic significance are not known. Do these changes in cyclic nucleotides have effects on epidermal proliferation? And does EGF? Agents which increase cyclic AMP do inhibit the epidermal outgrowth and mitotic activity of explant cultures of pig skin. Cyclic GMP does increase outgrowth at a particular concentration. Histamine, which elevates both cyclic nucleotides, has a biphasic action depending on its concentration. These findings imply that these nucleotides do act as one of the controls of epidermal proliferation. The action of cyclic GMP is not accompanied by detectably increased phosphorylation of epidermal proteins. On the other hand, EGF action which also enhances epidermal outgrowth is characterized by an increased protein phosphorylation that precedes any increase in cellular cyclic

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclases; Animals; Cell Division; Cyclic AMP; Cyclic GMP; Enzyme Activation; Epidermal Growth Factor; Epidermis; Histamine; Humans; Ischemia; Phosphoproteins; Protein Kinases; Swine

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Blood Glucose; Brain; Cyclic AMP; Cyclic GMP; Energy Metabolism; Gerbillinae; Glucose; Glycogen; Ischemia; Kinetics; Lactates; Phosphocreatine

By use of highly sensitive radioimmunoassays, 3':5'-cyclic AMP (cAMP) and 3':5'-cyclic GMP (cGMP) were measured in individual layers of light- and dark-adapted rabbit retinas, and the effects of ischemia were determined. In light-adapted retinas, cGMP levels ranged 50-fold, with over 90% of the total concentrated in the photoreceptor cells. The layer of outer segments contained 95 mumol/kg of dry weight, or three times the concentration present in the remainder of the photoreceptor cell layers. By contrast, levels of cAMP varied only 4-fold; the lowest level (6 mumol/kg of dry weight) was found in the outer segment layer and the highest level (22 mumol/kg of dry weight) in the inner segment layer of the photoreceptor cells. Dark adaptation elevated cGMP levels only in retinal layers containing photoreceptor cells, and the greatest proportional increase was observed in the synaptic layer of photoreceptor cells. Dark adaptation also caused increases of cAMP that were restricted to the outer plexiform and outer nuclear layers. Ischemia lowered cGMP levels, but only in retinal layers containing photoreceptor cells, and elevated cAMP levels, primarily in the inner layers of the retina. The effects of ischemia were greater in the dark-adapted than in light-adapted retinas. These results indicate that cGMP and cAMP levels in retina are influenced by the light adaptational state, that ischemia markedly modifies these processes, and that the effects of both light exposure and ischemia are regionally selective.

Topics: Animals; Cyclic AMP; Cyclic GMP; Dark Adaptation; Ischemia; Light; Photoreceptor Cells; Rabbits; Retina; Retinal Vessels

Topics: Adenosine Triphosphate; Animals; Cerebral Cortex; Citrates; Cyclic GMP; Dopamine; Energy Metabolism; gamma-Aminobutyric Acid; Gerbillinae; Glutamates; Ischemia; Neurotransmitter Agents; Norepinephrine; Nucleotides; Phosphocreatine; Serotonin

Topics: Animals; Brain; Brain Stem; Cerebellum; Cerebral Cortex; Cyclic AMP; Cyclic GMP; Female; Freezing; Hippocampus; Hypothalamus; Ischemia; Male; Mice; Radioimmunoassay; Spinal Cord; Thalamus; Time Factors