Effect of nitric oxide reduction on arterial thrombosis.

OBJECTIVES: Nitric oxide (NO) represents the most powerful endogenous molecule with vasodilator action mainly produced by endothelial nitric oxide synthase (eNOS) enzyme. Polymorphisms and epigenetic-sensitive mechanisms can modulate the expression of eNOS gene, leading to the endothelial dysfunction. This review updates on the mechanistic role of NO in the regulation of platelet activation, as well as the impact of eNOS genetic and epigenetic modifications on arterial thrombosis onset. DESIGN: A systematic search was addressed to examination of PubMed databases with the following terms: nitric oxide; arterial thrombosis; endothelial dysfunction; DNA variations; epigenetic modifications; personalized therapy; network medicine. RESULTS: G894T, -786T/C, and 4b/4a variable number tandem repeat (VNTR), are the main classes of polymorphisms harbored in eNOS gene associated to increased arterial thrombosis risk. DNA methylation, histone/non-histone modifications, and microRNA (miRNAs) can modulate eNOS gene expression. Investigators largely focused on the role of miRNAs in modulating NO production in arterial thrombosis development. In detail, miR-195, and miR-582 are inversely correlated both to eNOS and NO levels, thus suggesting novel biomarkers. CONCLUSION: We are far from incorporating omics pathogenic data from bench to arterial thrombosis bedside. Network medicine is an emerging paradigm that ideally overcomes the current shortcomings of the reductionist approach. Despite several clinical limitations, the network-based analysis of the interactome might reveal the key nodes underlying the perturbations of the arterial thrombosis, thus advancing personalized therapy.

Transfusion of Platelets Loaded With Recombinant ADAMTS13 (A Disintegrin and Metalloprotease With Thrombospondin Type 1 Repeats-13) Is Efficacious for Inhibiting Arterial Thrombosis Associated With Thrombotic Thrombocytopenic Purpura.

Objective- ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) cleaves VWF (von Willebrand factor). This process is essential for hemostasis. Severe deficiency of plasma ADAMTS13 activity, most commonly resulting from autoantibodies against ADAMTS13, causes thrombotic thrombocytopenic purpura. Therapeutic plasma exchange is the standard of care to date, which removes autoantibodies and replenishes ADAMTS13. However, such a therapy is often ineffective to raise plasma ADAMTS13 activity, and in-hospital mortality rate remains as high as 20%. Approach and Results- To overcome the inhibition by autoantibodies, we developed a novel approach by delivering rADAMTS13 (recombinant ADAMTS13 ) using platelets as vehicles. We show that both human and murine platelets can uptake rADAMTS13 ex vivo. The endocytosed rADAMTS13 within platelets remains intact, active, and is stored in α-granules. Under arterial shear (100 dyne/cm2), the rADAMTS13 in platelets is released and effectively inhibits platelet adhesion and aggregation on a collagen-coated surface in a concentration-dependent manner. Transfusion of rADAMTS13-loaded platelets into Adamts13-/- mice dramatically reduces the rate of thrombus formation in the mesenteric arterioles after FeCl3 injury. An ex vivo transfusion of rADAMTS13-loaded platelets to a reconstituted whole blood containing plasma from a patient with immune-mediated thrombotic thrombocytopenic purpura and the cellular components (eg, erythrocytes and leukocytes) from a healthy individual, as well as a fresh whole blood obtained from a patient with congenital or immune-mediated thrombotic thrombocytopenic purpura also dramatically reduces the rate of thrombus formation under arterial flow. Conclusions- Our results demonstrate that transfusion of rADAMTS13-loaded platelets may be a novel and potentially effective therapeutic approach for arterial thrombosis, associated with congenital and immune-mediated thrombotic thrombocytopenic purpura.

Oxidative Stress in Aortas of Patients with Advanced Occlusive and Aneurysmal Diseases.

BACKGROUND: Aortoiliac occlusive disease (AOD) and abdominal aortic aneurysm (AAA) are very important cardiovascular diseases that present different aspects of pathophysiology; however, oxidative stress and inflammatory response seem be relevant in both of them. Our objective was to evaluate oxidative damage and degree of inflammatory infiltrate in aortas of patients surgically treated for AOD and AAA. MATERIALS AND METHODS: Levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and myeloperoxidase (MPO) expression as well as nitrite levels and superoxide dismutase (SOD) and catalase (CAT) activities were evaluated in aortas of patients with AOD (n = 16) or AAA (n = 14), while the control group was formed by cadaveric organ donors (n = 10). We also analyzed the degree of inflammatory infiltrate in these aortas. RESULTS: There was an increase in ROS levels and NADPH oxidase activity in patients with AOD and AAA when compared with the control group, and the AOD group demonstrated higher ROS production and NADPH oxidase activity and also nitrite levels when compared with the AAA group (P < 0.001). On the other hand, an increase of SOD activity in the AOD group and CAT activity in the AAA group was observed. Inflammatory infiltrate and MPO expression were higher in the AOD group when compared with the control group (P < 0.05). CONCLUSIONS: Oxidative stress is relevant in both AOD and AAA, though AOD presented higher ROS levels and NADPH activity. Increased activities of antioxidant enzymes may be a compensatory phenomenon which occurs in aortas of patients with AOD and AAA. Perhaps, a relationship between oxidative stress and degree of inflammatory infiltrate may exist in the pathophysiology of AOD and AAA.

Refractory Arterial Insufficiency Associated with Janus Kinase 2 Positive Essential Thrombocythaemia.

Essential thrombocythaemia (ET) is one of the severe rare clonal haematologic stem cell disorders that encompass myeloproliferative neoplasms. ET has a well-described association with peripheral arterial thrombosis, which presents a challenging clinical presentation. Further understanding into the underlying pathophysiology of thrombosis in ET has been made following the identification of the Janus Kinase 2 (JAK2) mutation, which is thought to confer a prothrombotic phenotype. Here we present a case of refractory arterial insufficiency associated with JAK2-positive ET.

The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.

Despite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, p<0.05). Similarly, human aortic endothelial cells exposed to cigarette smoke total particulate matter (CS-TPM) revealed increased TF expression mediated by JNK2 (n=4; p<0.05). Wild-type and JNK2-/- mice were exposed to cigarette smoke for two weeks after which arterial thrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; p<0.05) and increased tissue factor activity (n=7; p<0.05) as compared to wild-type controls (n=6), while JNK2-/-mice exposed to smoke maintained an unaltered thrombotic potential (n=8; p=NS) and tissue factor activity (n=8) comparable to that of JNK2-/- and wild-type controls (n=6; p=NS). Smoking caused an increased production of reactive oxygen species (ROS) in wild-type but not in JNK2-/- mice (n=7; p<0.05 for wild-type mice and n=5-6; p=NS for JNK2-/- mice). In conclusion, the MAP kinase JNK2 mediates cigarette smoke-induced TF activation, arterial thrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.

Oxidative stress in human aorta of patients with advanced aortoiliac occlusive disease

Abstract Introduction: Oxidative stress seems to be a role in the atherosclerosis process, but research in human beings is scarce. Objective: To evaluate the role of oxidative stress on human aortas of patients submitted to surgical treatment for advanced aortoiliac occlusive disease. Methods: Twenty-six patients were divided into three groups: control group (n=10) formed by cadaveric organ donors; severe aortoiliac stenosis group (patients with severe aortoiliac stenosis; n=9); and total aortoiliac occlusion group (patients with chronic total aortoiliac occlusion; n=7). We evaluated the reactive oxygen species concentration, nicotinamide adenine dinucleotide phosphate-oxidase, superoxide dismutase and catalase activities as well as nitrite levels in samples of aortas harvested during aortofemoral bypass for treatment of advanced aortoiliac occlusive disease. Results: We observed a higher level of reactive oxygen species in total aortoiliac occlusion group (48.3±9.56 pmol/mg protein) when compared to severe aortoiliac stenosis (33.5±7.4 pmol/mg protein) and control (4.91±0.8 pmol/mg protein) groups (P<0.05). Nicotinamide adenine dinucleotide phosphate oxidase activity was also higher in total aortoiliac occlusion group when compared to the control group (3.81±1.7 versus 1.05±0.31 µmol/min.mg protein; P<0.05). Furthermore, superoxide dismutase and catalase activities were significantly higher in the severe aortoiliac stenosis and total aortoiliac occlusion groups when compared to the control cases (P<0.05). Nitrite concentration was smaller in the severe aortoiliac stenosis group in comparing to the other groups. Conclusion: Our results indicated an increase of reactive oxygen species levels and nicotinamide adenine dinucleotide phosphate-oxidase activity in human aortic samples of patients with advanced aortoiliac occlusive disease. The increase of antioxidant enzymes activities may be due to a compensative phenomenon to reactive oxygen species production mediated by nicotinamide adenine dinucleotide phosphate oxidase. This preliminary study offers us a more comprehensive knowledge about the role of oxidative stress in advanced aortoiliac occlusive disease in human beings.

Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice.

OBJECTIVE: Circulating blood cells and endothelial cells express ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. APPROACH AND RESULTS: To determine how CD73 activity influences in vivo thrombosis, the time to ferric chloride-induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl3, but not to 10% FeCl3, there was a significant decrease in the time to thrombosis in CD73-null mice compared with wild-type mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor α-ß-methylene-ADP nullified the prolongation in the time to thrombosis in human CD39 transgenic (hC39-Tg)/CD73-null mice. To determine whether hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl3-induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted wild-type mice, but not on wild-type reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared with hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. CONCLUSIONS: These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis.

The AST/ALT (De-Ritis) ratio: A novel marker for critical limb ischemia in peripheral arterial occlusive disease patients.

The aspartat aminotransferase (AST)/alanin aminotransferase (ALT) (De-Ritis) ratio (AAR) is an easily applicable blood test. An elevated AAR on the one hand has been associated with an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD on the other hand is associated with an increase in cardiovascular disease, all-cause mortality, and diabetes. As the AAR is also elevated in case of muscular damage, we investigated AAR and its association with critical limb ischemia (CLI) in peripheral arterial occlusive disease (PAOD) patients.In our cross-sectional study, we included 1782 PAOD patients treated at our institution from 2005 to 2010. Patients with chronic alcohol consumption (>20 g/day) were excluded. AAR was calculated and the cohort was categorized into tertiles according to the AAR. An optimal cut-off value for the continuous AAR was calculated by applying a receiver operating curve analysis to discriminate between CLI and non-CLI.In our cohort, occurrence of CLI significantly increased with an elevation in AAR. As an optimal cut-off value, an AAR of 1.67 (sensitivity 34.1%, specificity 81.0%) was identified. Two groups were categorized, 1st group containing 1385 patients (AAR < 1.67) and a 2nd group with 397 patients (AAR > 1.67). CLI was more frequent in AAR > 1.67 patients (166 [41.9%]) compared to AAR < 1.67 patients (329 [23.8%]) (P < 0.001), as was prior myocardial infarction (28 [7.1%] vs 54 [3.9%], P = 0.01). Regarding inflammatory parameters, C-reactive protein (median 8.1 mg/L [2.9-28.23] vs median 4.3 mg/L [2.0-11.5]) and fibrinogen (median 427.5 mg/dL [344.25-530.0] vs 388.0 mg/dL [327.0-493.0]) also significantly differed in the 2 patient groups (both P < 0.001). Finally, an AAR > 1.67 was associated with an odds ratio (OR) of 2.0 (95% confidence interval [CI] 1.7-2.3) for CLI even after adjustment for other well-established vascular risk factors.An increased AAR is significantly associated with patients at high risk for CLI and other cardiovascular endpoints. The AAR is a broadly available and cheap marker, which might be useful to highlight patients at high risk for vascular endpoints.

The Novel Oral Syk Inhibitor, Bl1002494, Protects Mice From Arterial Thrombosis and Thromboinflammatory Brain Infarction.

OBJECTIVE: Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is the second leading cause of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein VI (GPVI) is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of not only GPVI but also other platelet and immune cell receptors. We sought to assess whether Syk might be an effective antithrombotic target. APPROACH AND RESULTS: We demonstrate that mice lacking Syk in platelets specifically are protected from arterial thrombus formation and ischemic stroke but display unaltered hemostasis. Furthermore, we show that mice treated with the novel, selective, and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 hours after transient middle cerebral artery occlusion, also when BI1002494 was administered therapeutically, that is, after ischemia. CONCLUSIONS: These results provide direct evidence that pharmacological Syk inhibition might provide a safe therapeutic strategy to prevent arterial thrombosis and to limit infarct progression in acute stroke.

Oxidative Stress in Human Aorta of Patients with Advanced Aortoiliac Occlusive Disease.

Introduction: Oxidative stress seems to be a role in the atherosclerosis process, but research in human beings is scarce. Objective: To evaluate the role of oxidative stress on human aortas of patients submitted to surgical treatment for advanced aortoiliac occlusive disease. Methods: Twenty-six patients were divided into three groups: control group (n=10) formed by cadaveric organ donors; severe aortoiliac stenosis group (patients with severe aortoiliac stenosis; n=9); and total aortoiliac occlusion group (patients with chronic total aortoiliac occlusion; n=7). We evaluated the reactive oxygen species concentration, nicotinamide adenine dinucleotide phosphate-oxidase, superoxide dismutase and catalase activities as well as nitrite levels in samples of aortas harvested during aortofemoral bypass for treatment of advanced aortoiliac occlusive disease. Results: We observed a higher level of reactive oxygen species in total aortoiliac occlusion group (48.3±9.56 pmol/mg protein) when compared to severe aortoiliac stenosis (33.5±7.4 pmol/mg protein) and control (4.91±0.8 pmol/mg protein) groups (P<0.05). Nicotinamide adenine dinucleotide phosphate oxidase activity was also higher in total aortoiliac occlusion group when compared to the control group (3.81±1.7 versus 1.05±0.31 µmol/min.mg protein; P<0.05). Furthermore, superoxide dismutase and catalase activities were significantly higher in the severe aortoiliac stenosis and total aortoiliac occlusion groups when compared to the control cases (P<0.05). Nitrite concentration was smaller in the severe aortoiliac stenosis group in comparing to the other groups. Conclusion: Our results indicated an increase of reactive oxygen species levels and nicotinamide adenine dinucleotide phosphate-oxidase activity in human aortic samples of patients with advanced aortoiliac occlusive disease. The increase of antioxidant enzymes activities may be due to a compensative phenomenon to reactive oxygen species production mediated by nicotinamide adenine dinucleotide phosphate oxidase. This preliminary study offers us a more comprehensive knowledge about the role of oxidative stress in advanced aortoiliac occlusive disease in human beings.

Proteolytically active ADAM10 and ADAM17 carried on membrane microvesicles in human abdominal aortic aneurysms.

The intraluminal thrombus (ILT) of human abdominal aortic aneurysm (AAA) has been suggested to damage the underlying aortic wall, but previous work found scant activity of soluble proteases in the abluminal layer of the ILT, adjacent to the aneurysm. We hypothesised that transmembrane proteases carried by membrane microvesicles (MV) from dying cells remain active in the abluminal ILT. ILTs and AAA segments collected from 21 patients during surgical repair were assayed for two major transmembrane proteases, ADAM10 (a disintegrin and metalloprotease-10) and ADAM17. We also exposed cultured cells to tobacco smoke and assessed ADAM10 and ADAM17 expression and release on MVs. Immunohistochemistry showed abundant ADAM10 and ADAM17 protein in the ILT and underlying aneurysmal aorta. Domain-specific antibodies indicated both transmembrane and shed ADAM17. Importantly, ADAM10 and ADAM 17 in the abluminal ILT were enzymatically active. Electron microscopy of abluminal ILT and aortic wall showed MVs with ADAM10 and ADAM17. By flow cytometry, ADAM-positive microvesicles from abluminal ILT carried the neutrophil marker CD66, but not the platelet marker CD61. Cultured HL60 neutrophils exposed to tobacco smoke extract showed increased ADAM10 and ADAM17 content, cleavage of these molecules into active forms, and release of MVs carrying mature ADAM10 and detectable ADAM17. In conclusion, our results implicate persistent, enzymatically active ADAMs on MVs in the abluminal ILT, adjacent to the aneurysmal wall. The production of ADAM10- and ADAM17-positive MVs from smoke-exposed neutrophils provides a novel molecular mechanism for the vastly accelerated risk of AAA in smokers.

Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad.

In a number of isolated blood vessel types, hypoxia causes an acute contraction that is dependent on the presence of nitric oxide and activation of soluble guanylyl cyclase. It is more pronounced when the preparations are constricted and is therefore termed hypoxic augmentation of vasoconstriction. This hypoxic response is accompanied by increases in the intracellular level of inosine 5'-triphosphate and in the synthesis of inosine 3',5'-cyclic monophosphate (cIMP) by soluble guanylyl cyclase. The administration of exogenous cIMP or inosine 5'-triphosphate causes augmented vasoconstriction to hypoxia. Furthermore, the vasoconstriction evoked by hypoxia and cIMP is associated with increased activity of Rho kinase (ROCK), indicating that cIMP may mediate the hypoxic effect by sensitizing the myofilaments to Ca through ROCK. Hypoxia is implicated in exaggerated vasoconstriction in the pathogenesis of coronary artery disease, myocardial infarction, hypertension, and stroke. The newly found role of cIMP may help to identify unique therapeutic targets for certain cardiovascular disorders.

Pelvic arterial occlusive disease affects the RhoA/Rho-kinase pathway in bladder smooth muscle.

PURPOSE: We investigated the effect of pelvic arterial occlusive disease on the RhoA/Rho-kinase pathway in a rat model of chronic bladder ischemia. MATERIALS AND METHODS: Male adult Sprague Dawley® rats at age 16 weeks were divided into arterial endothelial injury and control groups. The injury group underwent balloon endothelial injury of the bilateral iliac arteries and received a 2% cholesterol diet to induce pelvic arterial occlusive disease. The control group received a regular diet. At 8 weeks cystometrograms were performed. Bladder tissue was harvested for pharmacological studies and Western blot. RESULTS: Cystometrograms showed significantly lower bladder capacity in the arterial endothelial injury group than in controls. Organ bath studies revealed significantly decreased phasic contractions induced by carbachol in bladder strips from the injury group than from controls. In controls bladder strip tonic contractions induced by carbachol were significantly decreased compared with phasic contractions. However, no significant difference was observed between phasic and tonic contractions in the injury group. The Rho-kinase inhibitor Y-27632 produced a concentration dependent decrease in tonic contractions, which was more pronounced in the injury group. Western blot showed significantly increased RhoA and Rho-kinase ß expression in the injury group. CONCLUSIONS: Our results suggest that pelvic arterial occlusive disease can affect the RhoA/Rho-kinase pathway in the bladder. This pathway might possibly be involved in the maintenance of tonic contraction and contribute to the bladder hyperactivity caused by pelvic arterial occlusive disease.

Postconditioning protects skeletal muscle against a long-lasting vascular occlusion.

PURPOSE/AIM OF THE STUDY: Long-lasting lower limb arterial occlusion is a condition with high incidence and complication rates. With the absence of appropriate treatment to cure advanced complications, mortality rates are high. Postconditioning (PC) might be capable of limiting the degree of ischemic-reperfusion (IR) injuries, thus reducing complications and mortality rates. The aim of this study was to evaluate the impact of postconditioning during the first postoperative day on skeletal muscle after a long-lasting arterial occlusion. MATERIALS AND METHODS: Male Wistar rats (n = 72) underwent 8 hr of infrarenal aortic occlusion followed by 2, 6, 12, or 24 hr of reperfusion. In one group of each reperfusion period, postconditioning was applied. Muscle samples were collected for histological examinations. Furthermore, muscle fiber viability and muscle wet-to-dry ratio were assessed. Blood samples were taken for creatine-kinase measurements. RESULTS: Postconditioning strongly reduced morphological injury compared to the corresponding ischemic-reperfusion group (p < .001). Serum creatine-kinase levels showed a peak at 6 hr post-ischemia (IR: 6702.2 ± 797.5; PC: 5523.3 ± 769.3 IU/l) and decreased to normal level by the end of the experiment (Sham: 171.5 ± 71.6; IR: 186.2 ± 82.7; PC: 174.2 ± 72.4 IU/l). Creatine-kinase levels were significantly reduced by postconditioning (p2hr = .028; p6hr = .06; p12hr = .042). A marked decrease in viability was observed in the ischemic-reperfusion groups (2 hr: 11.0 ± 4.1; 6 hr: 10.3 ± 3.6; 12 hr: 9.4 ± 3.3; 24 hr: 8.6 ± 2.8%), whereas with postconditioning, viability was preserved (2 hr: 26.4 ± 5.5; 6 hr: 24.6 ± 4.5; 12 hr: 24.5 ± 6.8; 24 hr: 26.2 ± 6.1%; p < .001); moreover, a significant decrease in the wet-to-dry ratio was achieved (p < .001). CONCLUSION: Postconditioning was able to reduce local complications after a long-lasting lower limb vascular occlusion.

Heparanase regulates thrombosis in vascular injury and stent-induced flow disturbance.

OBJECTIVES: The purpose of this study was to examine the role of heparanase in controlling thrombosis following vascular injury or endovascular stenting. BACKGROUND: The use of endovascular stents are a common clinical intervention for the treatment of arteries occluded due to vascular disease. Both heparin and heparan sulfate are known to be potent inhibitors of thrombosis. Heparanase is the major enzyme that degrades heparan sulfate in mammalian cells. This study examined the role of heparanase in controlling thrombosis following vascular injury and stent-induced flow disturbance. METHODS: This study used mice overexpressing human heparanase and examined the time to thrombosis using a laser-induced arterial thrombosis model in combination with vascular injury. An ex vivo system was used to examine the formation of thrombus to stent-induced flow disturbance. RESULTS: In the absence of vascular injury, wild type and heparanase overexpressing (HPA Tg) mice had similar times to thrombosis in a laser-induced arterial thrombosis model. However, in the presence of vascular injury, the time to thrombosis was dramatically reduced in HPA Tg mice. An ex vivo system was used to flow blood from wild type and HPA Tg mice over stents and stented arterial segments from both animal types. These studies demonstrate markedly increased thromboses on stents with blood isolated from HPA Tg mice in comparison to blood from wild type animals. We found that blood from HPA Tg animals had markedly increased thrombosis when applied to stented arterial segments from either wild type or HPA Tg mice. CONCLUSIONS: Taken together, this study's results indicate that heparanase is a powerful mediator of thrombosis in the context of vascular injury and stent-induced flow disturbance.

Aging-induced collateral dysfunction: impaired responsiveness of collaterals and susceptibility to apoptosis via dysfunctional eNOS signaling.

Despite positive animal studies, clinical angiogenesis trials have been disappointing, possibly due to risk factors present in humans but usually unexplored in animals. We recently demonstrated aging causes impaired collateral remodeling and collateral dropout; here, we investigate potential mechanisms responsible for these findings. Four-, 10-, and 18-month-C57BL/6J mice were subjected to femoral artery ligation; flow was measured using laser Doppler perfusion imaging. Endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS were measured in calf muscle. Apoptosis was assessed in endothelial (EC) and smooth muscle (SMC) cells isolated from young and old mice. Angiogenesis was measured using a Matrigel plug assay. Lethally irradiated young and old mice received bone marrow cells (BMC) from either young or old donors and were subjected to femoral artery ligation (FAL). BMC mobilization and homing were assessed. Flow recovery was impaired and less eNOS and phosphorylated eNOS was present in older vs. young mice (p < 0.001 and p = 0.015, respectively). ECs and SMCs from older mice were more sensitive to an apoptotic stimulus, but were rescued by NO-enhancing drugs. In older mice, angiogenesis (Matrigel plug assay) was impaired, as was mobilization and homing of BM progenitor cells following FAL. Although both mobilization and homing improved when older mice received BMC transplantation from young donors, flow recovery failed to improve. Aging impairs BMC mobilization and homing, collateral responsiveness to angiogenic stimuli, and increases EC and SMC susceptibility to apoptosis via dysfunctional eNOS signaling. The latter could contribute to impaired remodeling and collateral dropout. These finding identify potential obstacles to therapeutic interventions in elderly patients.

Protein kinase C{delta} deficiency accelerates neointimal lesions of mouse injured artery involving delayed reendothelialization and vasohibin-1 accumulation.

OBJECTIVE: To use protein kinase C (PKC) δ-knockout mice to investigate the role of PKCδ in lesion development and to understand the underlying mechanism of the vascular disease. METHODS AND RESULTS: PKCδ functions as a signal transducer mediating several essential functions of cell proliferation and apoptosis. However, the effect of PKCδ on neointimal formation in wire-injured vessels is unknown. Three weeks after wire injury of femoral arteries, neointimal lesions were significantly increased in PKCδ(-/-) mice compared with PKCδ(+/+) animals. Immunohistochemical staining revealed that total numbers of smooth muscle cells and macrophages in the lesions of PKCδ(-/-) mice were markedly elevated without changing the ratio of these 2 cell types. To further elucidate the mechanisms of PKCδ-mediated increase in the lesion, an in vivo endothelial migration model was established to evaluate endothelial wound healing after wire injury. Data showed that reendothelialization of the injured vessel was markedly delayed in PKCδ(-/-) mice; this coincided with more severe intimal hyperplasia. Migration of endothelial cells cultivated from cardiac tissue was markedly reduced in the absence of PKCδ, whereas no difference in proliferation or apoptosis was detected. Inhibition of PKCδ activity or protein expression by small hairpin RNA (shRNA) in cultured endothelial cells confirmed the defective migratory phenotype. Interestingly, vasohibin-1, an antiangiogenesis protein, was elevated in endothelial cells derived from PKCδ(-/-) mice, which was mainly because of delayed protein degradation mediated by PKCδ. Downregulation of vasohibin-1 restored the migration rate of PKCδ(-/-) endothelial cells to a similar level as PKCδ(+/+) cells. CONCLUSIONS: PKCδ deficiency enhances neointimal formation, which is associated with delayed reendothelialization and involves increased cellular vasohibin-1 accumulation.

Reduction of PKCbetaII activity in smooth muscle cells attenuates acute arterial injury.

OBJECTIVE: The ubiquitous enzyme protein kinase C (PKC) has been linked to the pathogenesis of vascular injury, but the cell-specific and discrete functions of the betaII isoform have yet to be discovered in this setting. Our previous findings demonstrated significantly increased PKCbetaII in the membrane fraction of injured femoral arteries in wild type (WT) mice and revealed reduction of neointimal expansion in PKCbeta(-/-) mice after acute vascular injury. As PKCbeta(-/-) mice are globally devoid of PKCbeta, we established novel transgenic (Tg) mice to test the hypothesis that the action of PKCbetaII specifically in smooth muscle cells (SMCs) mediates the formation of neointimal lesions in response to arterial injury. METHODS: Tg mice expressing SM22alpha promoter-targeted mouse carboxyl-terminal deletion mutant PKCbetaII were produced using standard techniques, subjected to femoral artery injury and compared with littermate controls. Smooth muscle cells (SMCs) were isolated from wild type (WT) and Tg mice and exposed to a prototypic stimulus, tumor necrosis factor (TNF)-alpha. Multiple strategies were employed in vivo and in vitro to examine the molecular mechanisms underlying the specific effects of SMC PKCbetaII in neointimal expansion. RESULTS: In vivo and in vitro analyses demonstrated that PKCbetaII activity in SMCs was critical for neointimal expansion in response to arterial injury, at least in part via regulation of ERK1/2, Egr-1 and induction of MMP-9. CONCLUSIONS: These data identify the SMC-specific regulatory role of PKCbetaII in neointimal expansion in response to acute arterial injury, and suggest that targeted inactivation of PKCbetaII may be beneficial in limiting restenosis via suppression of the neointima-mediating effects of Egr-1 and MMP-9.

Hemin prevents in-stent stenosis in rat and rabbit models by inducing heme-oxygenase-1.

OBJECTIVE: The introduction of drug-eluting stents (DES) has largely added benefit to the percutaneous coronary intervention. Questions about the long-term safety of DES have been raised, however, particularly with respect to late stent thrombosis. Research efforts are now being directed toward therapeutics that can impede smooth muscle proliferation and promote vascular healing. Emerging data suggest that heme oxygenase-1 (HO-1), an inducible oxidoreductase enzyme system, can exert cytoprotective effects on endothelial cells and limit smooth muscle cell proliferation. We assessed the ability of hemin, a potent HO-1 inducer, to reduce in-stent stenosis without compromising re-endothelialization. METHODS: Rat aorta and rabbit iliac arteries were stented. Animals received ongoing treated with intraperitoneal hemin (50 mg/kg) or vehicle. At 7 to 28 days after surgery, stented arterial segments were collected and processed for histologic, electron microscopy, or protein analysis. RESULTS: In both models, treatment with hemin reduced neointima growth without compromising re-endothelialization of the stented arteries. In the rat aorta, analysis of protein expression at 7 and 28 days after stenting revealed that hemin increased HO-1 expression and limited the early inflammatory, apoptotic, and proliferative cellular events that are common to in-stent stenosis. Hemin treatment decreased the expression of the Ki-67 protein and the activity of key regulators of smooth muscle cell proliferation, including p42/44, RhoA, and up-regulated the expression of cyclin-dependent kinase inhibitors. The beneficial effects of hemin were abolished in the presence of tin-protoporphyrin IX, an HO inhibitor. Finally, treatment with tricarbonylchloro(glycinato)ruthenium(II), a carbon monoxide donor, reduced in-stent stenosis in the rat aorta, suggesting that carbon monoxide, a by-product of heme degradation, might contribute to the protective effect of hemin. CONCLUSION: These results suggest that HO-1 is important in limiting in-stent stenosis and can be regarded as a new therapeutic target.