Long term data of endovascularly treated patients with severe and complex aortoiliac occlusive disease.

AIM: This paper presents the recent data of the largest series (20 patients) of endovascularly treated patients and the first long term data of 9 patients with severe aortoiliac occlusive disease. METHODS: Between 2003 and 2012, 20 consecutive patients (14 men; 70 %) with Leriche syndrome underwent recanalization with solely endovascular means at our centre. The treatment strategy comprised the antegrade (transbrachial) recanalization of the occluded segments followed by retrograde (transfemoral) angioplasty with selective stent placement in the infrarenal aorta and primary stent placement in the iliac arteries. Before discharge, after 30 days and every year after the procedure, a clinical, as well as a duplex ultrasonographic examination including measurement of the ankle-brachial index was done. RESULTS: Bilateral success was achieved in 17 patients (85%). Unilateral success was achieved in three patients (15%). In one patient (5%) an early reocclusion of the stented segments occurred, necessitating bypass grafting. In nine patients long term data were evaluated. Here, the ankle brachial index (ABI) significantly increased (0.85 ± 0.15 vs. 0.51 ± 0.11 at baseline; P=0.002). Compared to baseline, the difference in the distribution of Rutherford category and the improvement of walking capacity were statistically significant (P=0.0006, P=0.01, respectively). CONCLUSION: This study shows the feasibility of solely endovascular management of severe aortoiliac occlusive disease with a high rate of success and low rate of complications. Significant clinical improvement of patients in long term follow up makes the endovascular approach a viable alternative to open surgery.

Myocardial infarction as complication of left atrial myxoma.

Although cardiac myxomas are histologically benign, they tend to form emboli and cause intracardiac obstruction, so that they must be classified as potentially fatal tumors of the heart. The probability of arterial embolism is closely correlated with the morphology of the tumor. Thus, villous myxomas are more fragile and form emboli more often. Nuclear spin tomography and echocardiographic cine-mode sequences provide impressive images of the potential for embolism. It appears that coronary embolism may be more frequent in the group of myxoma patients than generally is assumed. These may present as acute myocardial ischemia with the typical clinical symptoms of acute myocardial infarction, as a silent infarct, shock, syncope or as sudden cardiac death. Besides our case report this paper will give an overview on published data on coronary embolism in patients with atrial myxoma. Interestingly there is a tendency for spontaneous recanalization of the obstructed coronary vessels, perhaps because of the tumors' tissue composition. Therefore it is reasonable to perform transoesophageal echocardiography to check out embolic sources like myxoma, when pathogenesis of myocardial infarction remains unclear after coronary angiography.

Changes in caveolin subtype protein expression in aging rat organs.

It is now accepted that caveolin plays a key role in signal transduction by directly binding to and regulating the function of molecules involved in transmembrane signaling, such as ras, suggesting that the amount of caveolin within cells may be an important factor in determining the cellular signaling. We investigated the ontogenic changes in the protein amount of caveolin subtypes, as well as ras protein expression in various organs (the heart, lungs, and muscles) obtained from aging rats (neonates, young and old adults). Our results demonstrated that caveolin protein expression changed ontogenically in a subtype-dependent manner. In lungs, for example, caveolin-1 expression changed in an opposite manner to caveolin-3 expression, while in the heart caveolin-1 and -3 changed in parallel. Ras expression showed an ontogenic increase in lungs and a decrease in muscles, which were both parallel to caveolin-1 expression. Our results suggest that the regulation of transmembrane signaling by caveolin may differ among developmental stages and caveolin subtypes.

Desensitization of the pulmonary adenylyl cyclase system: a cause of airway hyperresponsiveness in congestive heart failure?

OBJECTIVES: This study was designed to investigate whether the adrenergic signal transduction in the lung and the responsiveness of airway smooth muscle to adrenergic stimulation are modulated in congestive heart failure. BACKGROUND: Wheezing and airway hyperresponsiveness are often present in heart failure. In the failing heart, chronic adrenergic stimulation down-regulates beta-adrenergic receptors and adenylyl cyclase. We hypothesized that airway dysfunction in heart failure could be due to a similar modulation of pulmonary adrenergic signal transduction. METHODS: Heart failure was induced in rats by aortic banding, resulting in increases in plasma norepinephrine, lung wet weight indicating congestion and left ventricular end diastolic pressure after four weeks. Beta-receptor densities in pulmonary plasma membranes were measured by radioligand binding using [125I]iodocyanopindolol. The G protein levels were determined by Western blot. Adenylyl cyclase activities in lung membranes were quantified as [32P]cAMP (cyclic adenosine-5'-monophosphate) synthesis rate. To functionally assess airway smooth muscle relaxation, carbachol-precontracted isolated tracheal strips were used. RESULTS: Beta-receptor density was significantly decreased in heart failure from 771 +/- 89 to 539 +/- 44 fmol/mg protein without changes in receptor affinities. The beta1-/beta2-subtype ratio, however, remained constant. The G(i and alpha) and G(s alpha) protein expression was unchanged. Adenylyl cyclase activity stimulated directly with forskolin was decreased by 28%. Relaxation of tracheal strips in response to isoproterenol and forskolin, but not to papaverin, was diminished by 30%. CONCLUSIONS: In heart failure, the down-regulation of pulmonary beta-receptors and concomitant decrease in adenylyl cyclase activity result in a significant attenuation of cAMP-mediated airway relaxation. These mechanisms may play a pivotal role in the pathogenesis of"cardiac asthma."

Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.

Downregulation of caveolin expression by cAMP signal.

Recent data have demonstrated that caveolin, a major structural protein of caveolae, inhibits the function of molecules involved in cAMP signaling such as adenylyl cyclase. We examined the effect of cAMP signal on the expressions of caveolin subtypes using rat cardiac myoblasts (H9C2 cells) and smooth muscle cells (RASMC), which express caveolin subtypes. Treatment of RASMC and H9C2 cells with forskolin, an adenylyl cyclase stimulator, decreased caveolin-1 mRNA levels in a dose-dependent manner. Time course studies showed a time-dependent decrease of caveolin-1 mRNA levels in H9C2 cells (after 6 hours) while caveolin-1 mRNA levels in RASMC showed a biphasic response, i.e., an initial increase (within 3 hours) and a later decrease (after 3 hours). Similar biphasic changes were observed when RASMC was treated with IBMX, a phosphodiesterase inhibitor. The levels of caveolin-1 and -3 proteins were also decreased by forskolin treatment, but only after 60-72 hours in RASMC and 24-36 hours in H9C2 cells. In contrast, the expression of caveolin-2 remained similar in both cells and decreased to a small degree after prolonged treatment. Therefore, the expression of caveolin is downregulated by cAMP signal in a caveolin subtype-dependent manner.

Forskolin derivatives with increased selectivity for cardiac adenylyl cyclase.

The current study was undertaken to examine whether we can target adenylyl cyclase to regulate beta-adrenergic signaling with increased cardiac selectivity. Forskolin, a natural diterpene compound, interacts directly with adenylyl cyclase. We studied the adenylyl cyclase isoform-selectivity of forskolin derivatives using insect cell membranes overexpressing type II, III, and V adenylyl cyclase isoforms. 6-[3-(dimethylamino) propionyl] forskolin (NKH477) stimulated type V more potently (1.87 +/- 0.02-fold) than type II (1.04 +/- 0.02-fold) and type III (0.89 +/- 0.03-fold) relative to forskolin (50 microM, P < 0.05). Similarly, 6-[3-(dimethylamino)propionyl]-14,15-dihydro-forskolin (DMAPD) stimulated type V (1.39 +/- 0.02-fold) more potently than types II (0.66 +/- 0.02-fold) and type III (0.31 +/- 0.02-fold) relative to forskolin (P < 0.05). This selectivity was maintained under different assay conditions--i.e. with different forskolin (0.1-100 microM) and Mg (1-10 mM) concentrations, with or without Gs alpha. NKH477 increased cAMP accumulation in HEK293 cells stably overexpressing type V more than forskolin (1.57 +/- 0.13-fold) (P < 0.05). Examination of multiple tissue homogenates revealed that DMAPD and NKH477 stimulated cardiac adenylyl cyclase more potently than the other tissue adenylyl cyclases (lung, brain, and kidney) relative to forskolin. Our results suggest that a particular side-chain modification of forskolin enhanced the selectivity for the cardiac isoform stimulation. Adenylyl cyclase isoforms may be targeted to increase tissue selectivity in future drug therapy for beta-adrenergic regulation.

Isoform-specific regulation of adenylyl cyclase by oxidized catecholamines.

Both epinephrine and manganese are known to stimulate cAMP production in cardiac homogenates. When added together, however, they inhibited adenylyl cyclase catalytic activity. Type V adenylyl cyclase, the major isoform in the heart, was also inhibited when an increasing concentration of epinephrine was added in the presence of manganese. Inhibition was not dependent on the condition of stimulation or preparation of the enzyme. However, this inhibition was abolished in the presence of anti-oxidant. Other catecholamines, including dopamine and isoproterenol, as well as adrenochrome, an oxidized product of epinephrine, similarly inhibited the activity of this enzyme. Kinetic analyses revealed that the K(m) for the substrate ATP was unchanged, but the V(max) was significantly decreased. In contrast, type II adenylyl cyclase, a non-cardiac isoform, was resistant to such inhibition by adrenochrome and was somewhat stimulated by it. Thus, catecholamines, when oxidized, directly interacted with adenylyl cyclase in an isoform-specific manner in the absence of G proteins. Our findings suggest that adenylyl cyclase isoforms have different sensitivity to various stresses, including oxidative stress.

Downregulation of caveolin by chronic beta-adrenergic receptor stimulation in mice.

Caveolae, flask-shaped invaginations of cell membranes, are believed to play pivotal roles in transmembrane transportation of molecules and cellular signaling. Caveolin, a structural component of caveolae, interacts directly with G proteins and regulates their function. We investigated the effect of chronic beta-adrenergic receptor stimulation on the expression of caveolin subtypes in mouse hearts by immunoblotting and Northern blotting. Caveolin-1 and -3 were abundantly expressed in the heart and skeletal muscles, but not in the brain. Continuous (-)-isoproterenol, but not (+)-isoproterenol, infusion via osmotic minipump (30 micrograms.g-1.day-1) for 13 days significantly downregulated both caveolin subtypes in the heart. The expression of caveolin-1 was reduced by 48 +/- 6.1% and that of caveolin-3 by 28 +/- 4.0% (P < 0.01, n = 8 for each). The subcellular distribution of caveolin subtypes in ventricular myocardium was not altered as determined by sucrose gradient fractionation. In contrast, the expression of both caveolin subtypes in skeletal muscles was not significantly changed. Our data suggest that the expression of caveolin subtypes is regulated by beta-adrenergic receptor stimulation in the heart.

Regulation of type V adenylyl cyclase by PMA-sensitive and -insensitive protein kinase C isoenzymes in intact cells.

Abstract Type V adenylyl cyclase (AC) was stably over-expressed in HEK293 cells (293AC-V). Forskolin-stimulated cAMP accumulation in 293AC-V was 5 times as great as that in control cells. PMA, a protein kinase C (PKC) activator, enhanced cAMP accumulation in 293AC-V cells dose-and time-dependently and this enhancement was abolished by staurosporine. Insulin also enhanced cAMP accumulation in 293AC-V cells. Co-transfection of PKC-zeta, but not PKC-alpha, potentiated the effects of insulin. These data suggest that type V AC activity is regulated in cells by PKC isoenzymes through different extracellular stimuli.

Impaired function of inhibitory G proteins during acute myocardial ischemia of canine hearts and its reversal during reperfusion and a second period of ischemia. Possible implications for the protective mechanism of ischemic preconditioning.

A brief antecedent period of myocardial ischemia and reperfusion can delay cellular injury during a subsequent ischemic condition. Recent observations suggest that this protective mechanism depends on the continued activation of adenosine A1 receptors and Gi proteins. During acute myocardial ischemia, sufficient amounts of adenosine for maximal activation of adenosine A1 receptors are released, independent of a preconditioning ischemia. Hence, the protective mechanism of ischemic preconditioning may not exclusively be explained by activation of adenosine A1 receptors. As a working hypothesis, an increased responsiveness of Gi proteins toward receptor-mediated activation, leading to an increased response of Gi-regulated effectors, was tested in this study. In 47 anesthetized dogs, ischemia was induced by proximal ligation of the left anterior descending coronary artery. Animals underwent either a single period of 5 minutes of ischemia (n = 9), a single period of 15 minutes of ischemia (n = 10), 5 minutes of ischemia followed by 15 minutes of reperfusion (n = 8), 15 minutes of ischemia followed by 60 minutes of reperfusion (n = 5), or 5 minutes of ischemia followed by 15 minutes of reperfusion and a second period of 5 minutes of ischemia (n = 15). Sarcolemmal membranes were prepared from the central ischemic area and from the posterior left ventricular wall, which served as the control. During ischemia, carbochol-stimulated GTPase decreased by 38% (control, 33.5 +/- 17.7; ischemia, 24.2 +/- 15 pmol.min-1.mg protein-1; n = 9; P < .001). The decrease in carbachol-stimulated GTPase activity was associated with a 45% decrease in carbachol-mediated inhibition of adenylyl cyclase (control, 28.9 +/- 2.4% maximal inhibition; ischemia, 15.1 +/- 2.6% maximal inhibition; n = 5; P < .001). Prolongation of the ischemic period to 15 minutes did not lead to a further reduction of the Gi-mediated signal transduction. The binding properties of muscarinic receptors were not affected by ischemia. Furthermore, as demonstrated by carbachol-stimulated binding of [gamma-35S]GTP to sarcolemmal membranes, high- and low-affinity binding sites for the muscarinic antagonist carbachol, the EC50 for carbachol-stimulated GTPase activity and the substrate dependency of the high-affinity GTPase, the interaction between muscarinic receptors and inhibitory G proteins, and GTP binding to G proteins were not altered (n = 14). Immunoblotting with alpha 1- and alpha 2-specific antibodies did not indicate a loss of Gi proteins during ischemia that could explain the reduced GTPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)