Endothelial beta3-adrenoceptors mediate vasorelaxation of human coronary microarteries through nitric oxide and endothelium-dependent hyperpolarization.

BACKGROUND: Coronary vessel tone is modulated in part by beta-adrenergic relaxation. However, the implication of specific beta-adrenoceptor subtypes and their downstream vasorelaxing mechanism(s) in human coronary resistance arteries is poorly defined. beta3-Adrenoceptors were recently shown to vasodilate animal vessels and are expressed in human hearts. METHODS AND RESULTS: We examined the expression and functional role of beta3-adrenoceptors in human coronary microarteries and their coupling to vasodilating nitric oxide (NO) and/or hyperpolarization mechanisms. The expression of beta3-adrenoceptor mRNA and protein was demonstrated in extracts of human coronary microarteries. Immunohistochemical analysis revealed their exclusive localization in the endothelium, with no staining of vascular smooth muscle. In contractility experiments in which videomicroscopy was used, the nonspecific beta-agonist isoproterenol and the beta3-preferential agonist BRL37344 evoked an approximately 50% relaxation of endothelin-1-preconstricted human coronary microarteries. Relaxations were blocked by the beta1/beta2/beta3-adrenoceptor antagonist bupranolol but were insensitive to the beta1/beta2-adrenoceptor antagonist nadolol, confirming a beta3-adrenoceptor-mediated pathway. Relaxation in response to BRL37344 was absent in human coronary microarteries devoid of functional endothelium. When human coronary microarteries were precontracted with KCl (thereby preventing vessel hyperpolarization), the relaxation to BRL37344 was reduced to 15.5% and totally abrogated by the NO synthase inhibitor L-omega-nitroarginine, confirming the participation of a NO synthase-mediated relaxation. The NO synthase-independent relaxation was completely inhibited by the Ca2+-activated K+ channel inhibitors apamin and charybdotoxin, consistent with an additional endothelium-derived hyperpolarizing factor-like response. Accordingly, membrane potential recordings demonstrated vessel hyperpolarization in response to beta3-adrenoceptor stimulation. CONCLUSIONS: Beta3-adrenoceptors are expressed in the endothelium of human coronary resistance arteries and mediate adrenergic vasodilatation through both NO and vessel hyperpolarization.

Destruction of contrast microbubbles by ultrasound: effects on myocardial function, coronary perfusion pressure, and microvascular integrity.

BACKGROUND: Recent experimental data indicate that ultrasound-induced destruction of ultrasound contrast microbubbles can cause immediate rupture of the microvessels in which these microbubbles are located. METHODS AND RESULTS: To examine the functional and morphological significance of these findings in the heart, isolated rabbit hearts were perfused retrogradely with buffer containing ultrasound contrast agents and were insolated at increasing levels of acoustic energy with a broadband transducer emitting at 1.8 MHz and receiving at 3.6 MHz and operated in the triggered mode (1 Hz). At the end of each experiment, the hearts were fixed in glutaraldehyde and examined with light microscopy. Neither exposure to ultrasound alone or to contrast alone affected left ventricular developed pressure. By contrast, simultaneous exposure to contrast and ultrasound resulted in a reversible, transient mechanical index (MI)-dependent decrease in left ventricular developed pressure (to 83+/-5% of baseline at an MI of 1.6) and a transient MI-dependent increase in coronary perfusion pressure (to 120+/-6% of baseline at an MI of 1.6). Myocardial lactate release also showed significant increases with increasing MIs. Macroscopically, areas of intramural hemorrhage were identified over the beam elevation in hearts exposed to both contrast and high-MI ultrasound. Light microscopy revealed the presence of capillary ruptures, erythrocyte extravasation, and endothelial cell damage. The mean percentage of capillaries ruptured at an MI of 1.6 was 3.6+/-1.4%. CONCLUSIONS: Simultaneous exposure of isolated rabbit hearts to ultrasound and contrast agents results in an MI-dependent, transient depression of left ventricular contractile function, a rise in coronary perfusion pressure, an increase in lactate production, and limited capillary ruptures.

Insulin-like growth factor-I gene transfer by electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats.

Catabolic states caused by injury are characterized by a loss of skeletal muscle. The anabolic action of IGF-I on muscle and the reduction of its muscle content in response to injury suggest that restoration of muscle IGF-I content might prevent skeletal muscle loss caused by injury. We investigated whether local overexpression of IGF-I protein by gene transfer could prevent skeletal muscle atrophy induced by glucocorticoids, a crucial mediator of muscle atrophy in catabolic states. Localized overexpression of IGF-I in tibialis anterior (TA) muscle was performed by injection of IGF-I cDNA followed by electroporation 3 d before starting dexamethasone injections (0.1 mg/kg.d sc). A control plasmid was electroporated in the contralateral TA muscle. Dexamethasone induced atrophy of the TA muscle as illustrated by reduction in muscle mass (403 +/- 11 vs. 461 +/- 19 mg, P < 0.05) and fiber cross-sectional area (1759 +/- 131 vs. 2517 +/- 93 mum(2), P < 0.05). This muscle atrophy was paralleled by a decrease in the IGF-I muscle content (7.2 +/- 0.9 vs. 15.7 +/- 1.4 ng/g of muscle, P < 0.001). As the result of IGF-I gene transfer, the IGF-I muscle content increased 2-fold (15.8 +/- 1.2 vs. 7.2 +/- 0.9 ng/g of muscle, P < 0.001). In addition, the muscle mass (437 +/- 8 vs. 403 +/- 11 mg, P < 0.01) and the fiber cross-sectional area (2269 +/- 129 vs. 1759 +/- 131 mum(2), P < 0.05) were increased in the TA muscle electroporated with IGF-I DNA, compared with the contralateral muscle electroporated with a control plasmid. Our results show therefore that IGF-I gene transfer by electroporation prevents muscle atrophy in glucocorticoid-treated rats. Our observation supports the important role of decreased muscle IGF-I in the muscle atrophy caused by glucocorticoids.

Pathology of unstable plaque: correlation with the clinical severity of acute coronary syndromes.

OBJECTIVES: The aim of this study was to relate the various clinical presentations of acute coronary syndromes to the underlying plaque morphology as assessed from histopathologic analysis of plaque fragments obtained by directional coronary atherectomy (DCA). BACKGROUND: Autopsy studies have shown that unstable angina and infarction are related to plaque instability and involve events such as fissure or rupture of the fibrous cap, thrombosis and inflammation. The clinical severity and prognosis of acute coronary syndromes can be estimated by the Braunwald classification of unstable angina. Whether plaque morphology can be related to the Braunwald classification has not been evaluated. METHODS: Plaque fragments were obtained by DCA in 75 patients: 38 with unstable angina, 19 with stable angina and 18 with no symptoms after infarction. The presence of fibrous tissue, thrombus, high cellularity, inflammatory cells, atheroma, neovessels and "stellar-shaped" smooth muscle cells was evaluated in 7-micron thick sections by appropriate staining. The patients were classified according to clinical presentation without knowledge of the results of pathologic examination, and a plaque instability score was assigned. The risk of further cardiac events was classified as low, medium or high. RESULTS: Increasing severity of the score of unstable angina was associated with increasing prevalence of thrombus, high cellularity, atheroma and neovessels. Plaque from patients with unstable angina considered to be at low risk of further events appeared very similar to that of patients with stable angina, whereas the specific morphologic characteristics of plaque instability were more frequently observed as the clinical score and the risk of further events increased. After thrombolyzed infarction, plaque morphology depends on the delay between the acute event and DCA. Within 1 week after infarction, plaque still showed the morphologic characteristics of instability, whereas late DCA provided samples with morphologic features similar to those observed in patients with stable angina. CONCLUSIONS: The morphologic features of plaque fragments vary at different stages of acute coronary disease. The specific features of plaque instability correlate with the clinical scoring system of the Braunwald classification.

Expression of inducible nitric oxide synthase in human coronary atherosclerotic plaque.

OBJECTIVE: Macrophages in atherosclerotic plaque may express the inducible isoform of NO synthase (iNOS), which produces large amounts of NO. On one hand, the production of NO can be protective by its vasodilatory, antiaggregant and antiproliferative effects. On the other hand, the formation of peroxynitrite from NO may favour vasospasm and thrombogenesis. In this study, we investigated whether iNOS is present in human coronary atherosclerotic plaque, and we correlated these data with the clinical instability of the patients. METHODS: Fragments were retrieved by coronary atherectomy from 24 patients with unstable angina and 12 patients with stable angina. The presence of macrophages, and the production of TNF alpha, iNOS and nitrotyrosine were detected by immunocytochemistry. RESULTS: Macrophage clusters were found in 67% of stable patients and 87% of patients with unstable angina (NS). TNF alpha was expressed in about 50% of cases in both groups. iNOS was not expressed in fragments from stable patients but was found in macrophages from 58% of unstable patients (P < 0.001). The expression of iNOS was associated with the presence of nitrotyrosine residues, a marker of peroxynitrite formation. Expression of iNOS was correlated both with complaints of angina at rest (P < 0.05) and with the presence of thrombus at morphological examination (P < 0.001). CONCLUSION: The expression of iNOS may be induced in human coronary atherosclerotic plaque and is associated with different factors of instability.

Basic anatomical and physiological differences between species should be considered when choosing combinations for use in models of hepatic xenotransplantation: an investigation of the guinea pig-to-rat combination.

Published data on the guinea pig-to-rat hepatic xenotransplant model describe problems concerning poor graft reperfusion. To further investigate this phenomenon, orthotopic liver xenotransplantation between weight-matched guinea pigs and rats were performed using Kamada's technique. On reperfusion, all cases had portal venous inflow block with hypoperfusion of the hepatic parenchyma. Histological examination showed no evidence of hyperacute rejection, although deposits of IgG2a and C3 but not IgM were identified within the central area of the liver. To increase blood inflow, arterialized partial liver grafts were performed without changing the outcome. We hypothesize that the hypoperfusion may be related to anatomical and physiological differences between the species. Guinea pig portal vein branches were found to have muscular walls susceptible to spasm, and portal blood flow is four times greater in the guinea pig than in the rat because the guinea pig intestine is both longer (two times as long) and of greater diameter. The combination of reperfusion injury, early immunological events, and the rat's lower portal blood flow induces spasm of the intrahepatic portal system resulting in hypoperfusion. These findings demonstrate the importance of recognizing basic anatomical and physiological differences between species when selecting xenotransplantation models.

Effect of graft preservation and IgM depletion on guinea pig to rat cardiac xenograft survival.

BACKGROUND: Heterotopic guinea pig (GP) cardiac xenografts (XG) are hyperacutely rejected within minutes when transplanted into rats. METHODS: In this GP to rat cardiac XG model, we studied the effect on graft survival of a short cold preservation time (1 hr at 4 degrees C) in the presence or absence of rat anti-GP IgM preformed antibodies. The complete depletion of circulating IgM was obtained by two intraperitoneal injections of anti-rat IgM monoclonal antibody (MARM-4) on preoperative days -3 and -1. RESULTS: When the GP cardiac XG was cold preserved for 1 hr before transplantation, the mean graft survival time (MST) was 13.5+/-2.8 min, whereas without previous cold preservation, the MST was significantly prolonged to 51.5+/-12.3 min (P<0.001). Interestingly, the complete depletion of preformed circulating IgM before grafting significantly prolonged the MST of a cold-preserved XG to 37.1+/-11.3 min in comparison with a nondepleted recipient of a cold-preserved XG (P<0.02), but did not prolong the graft survival of a XG that was not cold preserved (42.5+/-14.1 min). To assess the effect of cold preservation and/or ischemia reperfusion, we intravenously injected a superoxide-dismutase mimetic (EUK-134) just before transplantation of a cold-preserved XG. This antioxidant regimen improved the MST from 13.5+/-2.8 min to 35.3+/-7.3 min (P<0.001). These results clearly suggested that either preservation lesions or preformed IgM are capable of accelerating the loss of the cardiac graft function, but also that the presence of preformed IgM seems to be especially deleterious when the cardiac XG has previously been ischemically injured. Analyzing the histological data, we also observed that the prompt cessation of cardiac function seen in cold-preserved grafts was uniformly associated with massive interstitial hemorrhage, thereby suggesting a particular susceptibility of the GP cardiac XG to cold preservation. To assess the effect of preservation on the GP cardiac function in a nonimmunological model, we performed syngeneic GP cardiac grafts and found that 1 hr of cold preservation provoked massive interstitial hemorrhage capable of promptly inducing the cessation of the heartbeat. CONCLUSIONS: Overall, this study demonstrated that both ischemic lesions and immunological processes might induce the cessation of cardiac graft function in the GP to rat model and this cessation of graft function is probably often misinterpreted as a XG rejection only.

Administration of exogenous interleukin-2 abrogates spontaneous rat liver allograft acceptance but does not affect long-term established graft survival.

BACKGROUND: Spontaneous tolerance to the orthotopic liver allograft uniformly occurs in the DA (RT1a) to PVG (RT1c) rat combination despite a fully allogeneic barrier. METHODS: To assess whether spontaneous acceptance might be the consequence of a T cell help deficit at the time of the first exposure of alloantigens to the host, we studied the effect of exogenous interleukin (IL)-2 injections at the time of liver transplantation and during long-term follow-up. RESULTS: Although spontaneous acceptance of the liver allograft constantly ensued in the DA to PVG combination, a daily injection of recombinant IL-2 (3 x 10(5) U) uniformly provoked acute cellular rejection of the liver allograft and consequently the death of animals by postoperative day 5-6. Simultaneous to the graft loss, hepatic enzymes (alanine aminotransferase) increased more than 50-fold in IL-2-treated recipients, whereas similar IL-2 treatment did not produce any hepatic dysfunction in syngeneic animals. By immunohistology, the expression of the alpha chain of the IL-2 receptor, usually undetectable in untreated animals, was evident on CD4 and CD8 lymphocytes infiltrating the liver graft. In contrast, a similar IL-2 regimen and even higher IL-2 doses (x 10(6) U) did not abrogate the liver allograft survival during long-term follow-up. CONCLUSIONS: Our results demonstrate that spontaneous rat liver allograft acceptance may be abolished by exogenous IL-2 injections, which suggests that an "inherent T cell help deficit" might be implicated in the spontaneous acceptance mechanisms of DA to PVG liver allografts.

In vivo IgM depletion by anti-mu monoclonal antibody therapy. The role of IgM in hyperacute vascular rejection of discordant xenografts.

Xenoreactive natural antibodies (XNA) and complement activation are thought to be the 2 main factors responsible for the hyperacute vascular rejection (HVR) of discordant xenografts. The aim of this work was to study the role of IgM XNA in the HVR of guinea pig to rat cardiac xenografts, a discordant model. Adult LOU/C rats were depleted of circulating IgM and therefore of IgM XNA using an anti-mu mAb (mouse anti-rat IgM mAb 7 [MARM-7]). Rats were injected with 10 mg and 5 mg of MARM-7 at days -3 and -1, respectively, and guinea pig cardiac xenografts were performed on day 0. Control animals were injected on the same days with 10 mg and 5 mg of anti-alpha mAb (MARA-1) or equivalent volumes of PBS. Xenografts were performed on day 0. Guinea pig cardiac xenograft survival time was significantly prolonged in IgM-depleted animals (62 min, P < 0.01) compared with controls using PBS (18 min) or MARA-1 mAb (12 min). This prolongation was not due to a decrease in the complement activity in IgM-depleted rats, since no significant variation of the C1q, C4, C3, and C5 complement hemolytic activity was observed between control and treated animals before HVR. Prolongation of the xenograft survival time in the MARM-7-treated group was correlated with an undetectable serum level of IgM and IgM XNA and a lack of IgM XNA deposits on the rejected xenograft vascular endothelium. Contrarily, both IgM-depleted and control animals showed C3 deposits on the rejected xenograft vascular endothelium and myocardium, as well as diffuse deposits of IgG2a XNA. Although HVR was not abrogated by the depletion of IgM XNA, our data indicate that IgM is implicated in the HVR and that the anti-mu approach is a potential therapeutic treatment for discordant xenografts. Finally, we suggest that other factors such as IgM-independent activation of complement might be one of the mechanisms responsible for the pathogenesis of HVR in the guinea pig to rat xenograft model.

Differential inhibition of B-cell development and xenoreactive natural antibody production by administration of anti-mu or anti-delta monoclonal antibodies in adult rats.

BACKGROUND: Given the role of xenoreactive natural antibodies (XNA) in the pathogenesis of xenograft rejection, we tested whether the administration of anti-mu or anti-delta monoclonal antibodies (mAbs) in adult rats would suppress the generation of XNA. METHODS: Adult LOU/C (Igkappa-1a) rats were treated with anti-mu or anti-delta mAbs after nonlethal total body irradiation and bone marrow transplantation from congenic LOU/C (Igkappa-1b) rats. The differentiation of donor bone marrow (BM)-driven Igkappa-1b+ B cells and XNA production were analyzed. RESULTS: Both anti-mu and anti-delta mAbs arrested B-cell differentiation in the BM. In anti-mu-treated rats, there was a total depletion of donor-driven, peripheral Igkappa-1b+ B cells, secreting cells, and circulating XNA of the Igkappa-1b allotype. In anti-delta-treated rats, a significant number of Igkappa-1b+ B cells, which did not express membrane IgD, "escaped" deletion and partially repopulated peripheral lymphoid organs. This B-cell population was active in the production of XNA, as revealed by the high serum levels of XNA in these animals. CONCLUSIONS: Anti-mu administration resulted in arrest of B-cell differentiation and in down-regulation of IgM and IgG XNA production in adult rats. These data suggest that the use of anti-mu mAbs may be a useful approach to suppress the production of XNA and prevent xenograft rejection. Furthermore, we suggest that the B-cell population responsible for the production of XNA in adult rats belongs to a B-cell lineage expressing low levels of membrane IgD and "escaping" deletion in the BM upon anti-delta treatment.

Differential effects of injections of anti-mu and anti-delta monoclonal antibodies on B-cell populations in adult mice: regulation of xenoreactive natural antibody-producing cells.

BACKGROUND: The depletion of differential B cell and xenoreactive natural antibodies (XNA) by anti-delta and anti-mu injections was analyzed in adult mice. Sequential treatment with anti-delta and then anti-mu induces a complete depletion of B cells and XNA and represents a potential approach to induce xenograft tolerance. METHODS: Adult mice were injected with anti-mu, anti-delta, anti-delta then anti-mu, or control isotype monoclonal antibodies from day 0 to day 14. The different B-cell populations were analyzed by FACS and immunohistology. Ig production was tested by ELISA. XNA were analyzed by FACS. RESULTS: Anti-mu injections induced a depletion of IgMhigh, immature B cells, marginal zone B cells, and B1 cells and an increase of IgG-XNA production. Anti-delta injections induced mature conventional IgDhigh B-cell depletion and increased IgM-XNA production. Interestingly, sequential injections of anti-delta then anti-mu induced a depletion of immature B cells, mature B cells (MZ, B2, and B1), and XNA. CONCLUSIONS: These results demonstrate that mature B-cell depletion in adult mice can be obtained by mAb injections and depends on the surface immunoglobulin cross-linking threshold. Indeed, anti-mu mAb depleted IgMhigh B cells (MZ and B1) and anti-delta, IgDhigh B cells (B2). The differential B-cell suppression shows that conventional B cells are responsible in the IgG-XNA production and MZ and B1 cells in the IgM-XNA production. Sequential repeated injections of anti-delta then anti-mu mAb depleted all B-cell populations and suppressed the whole XNA production.

Apoptosis of human naive NK cells mediated by a rat IgG2b anti CD2 mAb through a fractricidal ADCC reaction.

LO-CD2a/BTI-322, a rat anti human CD2 mAb, shows in vitro and in vivo immunosuppressive properties and induces T-cell depletion resulting partially from an antibody dependent cellular cytotoxicity (ADCC) mediated by NK cells. The aim of this paper is to study the in vitro effect of LO-CD2a/BTI-322 on NK cells, the majority of them also expressing the CD2 molecule. The addition of the mAb to purified naive NK cells induces apoptosis of CD2+ cells. The apoptosis is rapid, Fas ligand independent and completely inhibited by the calcium chelator EGTA, suggesting a fractricidal ADCC reaction and implying that NK cells are not resistant to lysis when used as target cells. At the end of the reaction, the CD2 - remaining cells are still capable of natural cytotoxicity against K562 cells, but at a lower rate than untreated cells.

Pathology of restenosis in saphenous bypass grafts after long-term stent implantation.

The implantation of saphenous vein grafts on the coronary arterial tree eventually leads to graft narrowing, which can be treated by the implantation of intravascular stents. However, long-term restenosis after stent implantation occurs in at least 30% of cases. Ten saphenous bypass grafts, in which a total of 12 stents had been implanted for an average of 32 months, were retrieved at least 10 months after implantation for angiographic diagnosis of reocclusion or severe restenosis. The metal struts were removed after macroscopic inspection of the vein, and the grafts were examined by light microscopy. Angiography revealed total occlusion in 9 stents and severe narrowing in 3. Pathologic examination revealed graft occlusion due to cellular hyperplasia in 4 cases and to recent thrombus formation in 5. Progression of atherosclerotic plaque was the cause of restenosis in the 3 severely narrowed grafts. In 2 of 5 grafts implanted with Palmaz-Schatz stents, the metallic struts had induced a local inflammatory reaction. Therefore, the long-term reocclusion of saphenous bypass grafts after stent implantation may be due to atherosclerotic plaque or fibromuscular hyperplasia. However, thrombus formation may still occur several years after implantation. In specific cases, stent implantation also induces inflammation around the stent struts.

Expression of endothelin-1 by adrenocortical carcinoma: a new target for anti-cancer therapy?

Adrenocortical carcinoma is a rare neoplasm with poor prognosis. Endothelin-1 (ET-1) has been implicated in carcinogenesis, but has never been studied in this neoplasm. A 76-year-old woman with Cushing's syndrome due adrenocortical carcinoma was operated on and the tumour removed was studied by immunohistochemistry for ET-1. Patient history illustrates the poor prognosis of this cancer that became metastatic after one year. Immunohistochemical studies disclosed a strong expression of ET-1 by adrenocortical carcinoma cells. As shown in other cancers, ET-1 expression by adrenocortical carcinoma may suggest a pathogenic role of ET-1 in tumorigenesis that possibly could be countered by ET-1 receptor antagonists. These agents could open new therapeutic perspectives to treat a carcinoma known to have a poor prognosis.