Inna Frolkis

Accelerated Fas-Mediated Apoptosis of Monocytes and Maturing Macrophages from Patients with Systemic Lupus Erythematosus: Relevance to In Vitro Impairment of Interaction with iC3b-Opsonized Apoptotic Cells

Impaired handling of apoptotic cells has been suggested as an important factor in the development of systemic lupus erythematosus (SLE), and a role for complement in the removal of apoptotic cells was shown recently. We studied the in vitro function of macrophages from 40 patients with SLE and their matched controls in the removal of heterologous apoptotic cells opsonized by iC3b. Interaction index of apoptotic cells opsonized by iC3b was significantly lower in patients with SLE and averaged 71% ± 37 of that of healthy individuals (p < 0.002) and 69% ± 35 of patients with rheumatoid arthritis (p < 0.007). SLE patients had increased apoptosis of both freshly isolated monocytes (p < 0.001) and maturing macrophages (p < 0.04) that led to decreased density of monocyte-derived macrophages. Apoptosis was inhibited by adding soluble Fas receptor indicating Fas-mediated apoptosis. As demonstrated in both healthy controls and patients with SLE, decreased macrophage density by itself caused significant decreased uptake of apoptotic cells by the remaining macrophages. Maintaining normal density in SLE patients either by an increased initial density or by using soluble Fas restored the interaction capacity of the individual macrophages in the majority of patients. We concluded that impaired in vitro interaction of iC3b-opsonized apoptotic cells with macrophages from patients with SLE was mainly associated with Fas-dependent accelerated apoptosis of the monocytes/macrophages. Accelerated apoptosis of phagocytes may represent a novel in vitro mechanism of impairment of interaction with apoptotic cells that, apart from reducing the number of professional phagocytes, alters the function of the remaining macrophages.

Vasoactive response of different parts of human internal thoracic artery to isosorbide-dinitrate and nitroglycerin: an in-vitro study

OBJECTIVEnThe left internal thoracic artery (LITA) is the most important graft for coronary artery bypass grafting (CABG). Its distal region is, however, prone to vasospasm. The effect of nitroglycerin (NTG) and isosorbide-dinitrate (ISDN) on different segments of this region was studied.nnnMETHODSnRings of three segments of the LITA were studied: 6-9 mm proximal to the bifurcation (part A); 1-3 mm proximal to the bifurcation (part B); and 3-6 mm distal to the bifurcation (part C). After baseline, maximal contraction of the rings was achieved using 60 mmol/l of KCl, they were exposed to increasing doses of ISDN and NTG (10-100 microg/ml), and dose-response curves were recorded.nnnRESULTSnThe contractile response of part A to KCl was significantly lower than that of parts B and C (1.87+/-0.25 versus 4.05+/-0.39 and 7.64+/-0.54 g, respectively; P<0.001). Both nitrates inhibited the contractile response in a concentration-dependent manner. The relaxing effects of both nitrates on part A was most pronounced (P<0.01), with the effect of ISDN being higher than that of NTG (P<0.01).nnnCONCLUSIONSnThe region 6-9 mm proximal to the LITA bifurcation is less prone to vasospasm, and has greater relaxation responses to ISDN and NTG than the more vasospastic distal parts of the LITA. We recommend avoiding the use of the very distal part of this artery during CABG, and to use high doses of ISDN rather than NTG as an anti-spastic measure.

Electromechanical impairment of human auricle and rat myocardial strip subjected to exogenous oxidative stress

OBJECTIVEnAnimal myocardial dysfunction induced by remote ischemia-reperfusion (IR) was shown to be partly accomplished via a direct effect of the pro-oxidant xanthine oxidase (XO). This direct remote effect was not tested in humans. We now assessed the performance of human auricles in the presence of solutions containing XO and/or allopurinol and compared them to those of rat myocardial strips.nnnMETHODSnHuman and rat specimens (n=64) were separately exposed for 2h to Krebs-Henseleit solution that either (1) exited from rat livers that were earlier perfused for 2h (control-human or control-rat), (2) exited from livers that were earlier made ischemic for 2h (IR-human, IR-rat), (3) contained xanthine (X) 3.8 microM + XO 3 mU ml(-1) (X+XO-human, X+XO-rat), or (4) exited from post 2h-ischemic livers and contained 100 microM allopurinol (human or rat IR + allopurinol groups).nnnRESULTSnUnlike the unchanged electromechanical performance in the control and IR+allopurinol auricles and strips, the rates of contraction, maximal force of contraction and working index of either preparation were reduced by 75-98% (P<0.01) when exposed to the IR reperfusate or to the X+XO-enriched Krebs. The basal amplitudes of contraction in these four latter groups increased twofold (P<0.01). XO activity was similarly low in the control and in the IR+allopurinol groups, but four- to 45-fold (P<0.001) higher in the IR and the X+XO groups, both in the rat and human organs. The reduced glutathione was reduced by approximately 50% (P<0.01) in either preparation in the IR and the X+XO groups compared to the control and IR+allopurinol groups.nnnCONCLUSIONSnRemotely and exogenously originated oxidative burst directly induces electromechanical dysfunction and disrupts oxidant/antioxidant balance in human auricles as it does in the rat myocardial strip.

Protamine Induces Vasorelaxation of Human Internal Thoracic Artery by Endothelial NO-Synthase Pathway

BACKGROUNDnProtamine is commonly used in cardiac surgery to reverse the anticoagulant effects of heparin. We investigated the role of different nitric oxide synthase pathways in the response of the human internal thoracic artery to protamine and evaluated whether heparin could prevent this effect.nnnMETHODSnA tension-recording method was used to obtain baseline measurements of contractions of human internal thoracic artery rings achieved with norepinephrine. Isolated internal thoracic artery rings were suspended in two organ chambers. One contained Krebs-Henseleit solution and served as control. The other contained a heparin or Nomega-Nitro-L-arginine (L-NAM, an inhibitor of both endothelial and inducible nitric oxide synthase) or a specific inhibitor of inducible nitric oxide synthase, aminoguanidine. Increasing doses of protamine were added to both chambers and dose-response curves were obtained.nnnRESULTSnProtamine was found to relax contracted internal thoracic arteries 56% +/- 4.7% of baseline measurements in a concentration-dependent manner. When L-NAM was added, protamine caused only a slight decrease of tension. There were no differences in the relaxing effect of protamine in the presence of aminoguanidine or heparin.nnnCONCLUSIONSnProtamine induces nitric oxide-dependent relaxation of the internal thoracic artery by activation of endothelial nitric oxide synthase pathway. Heparin could not prevent this relaxing effect of protamine.

Vipera aspis venom reduces lethality and down-regulates tumor necrosis factor-α in a rat model of LPS-induced sepsis

OBJECTIVESnSepsis and septic shock are major causes of morbidity and mortality in critically-ill patients. Sepsis constitutes the systemic response to infection, that is predominantly mediated by the pro-inflammatory cytokines TNF-alpha and IL-1beta. Hence, cytokine modulation provides a promising target for the treatment of sepsis. In this work we evaluated the effect of a low-dose Vipera aspis venom (VAV) vaccine on survival and cytokine serum levels in a rat model of lipopolysaccharide (LPS)-induced septic shock.nnnMETHODSnAdult male Wistar rats were given either VAV vaccine or saline, and 2 weeks later half of each group received LPS challenge, and were monitored for mortality, cytokine levels, blood count and chemistry.nnnRESULTSnSurvival rate was significantly higher in venom-treated, compared to non-vaccinated septic rats. Furthermore, VAV treatment significantly reduced LPS-associated TNF-alpha and LDH, without affecting IL-6 and IL-10 levels, and modified WBC and platelet counts.nnnCONCLUSIONSnOur data suggest that sub-toxic doses of VAV have a protective effect against LPS-induced septic shock that may be mediated, at least partially, by the modulated TNF-alpha activity. This study thus offers a novel therapeutic approach for the attenuation of bacteremia-induced septic shock through the modulation of a central pro-inflammatory cytokine by VAV vaccination in mammals.

Tumor necrosis factor as a mediator of cardiac toxicity following snake envenomation

ObjectiveTo investigate the possible role of tumor necrosis factor in mediating cardiotoxicity following venom injection in a rat. DesignA randomized controlled experimental study using a Langendorff isolated heart model. SettingAnimal laboratory. SubjectsAdult male Wistar rats. InterventionsThe control group (n = 10) was injected with saline only. Each animal in the experimental groups 1–3 (n = 10 each) was injected with Vipera aspis venom 500 &mgr;g/kg intramuscularly. Group 1 animals received no additional substance beforehand, group 2 animals were injected intramuscularly with 250 &mgr;g of soluble tumor necrosis factor receptor (sTNF-R p55) 15 mins before the venom injection, and group 3 animals were injected intraperitoneally with 40 &mgr;g of anti-tumor necrosis factor 60 mins before the venom injection. Measurements and Main ResultsCardiac performances were investigated following envenomation. Cardiac histology and myocardial tumor necrosis factor-RNA concentrations were assessed. Serum tumor necrosis factor concentrations rose and peaked 2 hrs following envenomation. A reduction in peak systolic pressures, maximum and minimum change in pressure over time, time-pressure integral, and coronary flow occurred in the venom-only-injected rats compared with controls, whereas blocking tumor necrosis factor activity prevented the deleterious cardiac effects of the envenomation. No histologic changes or increases in myocardial tumor necrosis factor-RNA concentrations were detected. ConclusionThese results strongly suggest that systemic release of tumor necrosis factor mediates cardiac toxicity following Vipera aspis envenomation.

Protamine cardiotoxicity and nitric oxide

OBJECTIVESnThe purpose of this study is to assess the role of the nitric oxide (NO) pathway in protamine-induced cardiotoxicity and to formulate a possible explanation for this adverse effect.nnnMETHODSnIsolated rat hearts were perfused by Krebs--Henseleit (KH) solution using a modified Langendorff model. They were randomized into three groups: A, 40 min perfusion with KH solution; B, 20 min perfusion with KH solution and 20 min with protamine; C, as B but Ng-monomethyl-L-arginine (L-NMMA), a non-selective inhibitor of the NO pathway, was added during 40 min of the perfusion period. Left ventricular (LV) function was measured every 10 min. NO and tumor necrosis factor-alpha (TNF) were detected in the effluent from the coronary sinus (CS) and in the supernatant of the cardiac myocytes culture. Nitric oxide synthases (NOS) mRNA levels were determined in groups A and B from LV samples at baseline and after 40 min of perfusion.nnnRESULTSnWe found that protamine at a dose of 12 microg/ml causes significant depression of LV function (decreased peak systolic pressure to 22.5+/-3.2% and dP/dt max to 22.9+/-3.1%). L-NMMA did not prevent protamine cardiotoxicity. NOS mRNA was not detected from LV samples in any group. The NO in the effluent from the CS and from the supernatant of the cardiomyocytes culture was below detectable levels. However, a significant amount of TNF was measured in the effluent from the CS (108+/-17 pg/min for group B and 117+/-13 pg/min for group C) and in the supernatant of the cardiomyocytes culture (65+/-21 pg/ml).nnnCONCLUSIONSnThis study suggests that direct protamine-induced cardiotoxicity does not depend on the NO pathway. Our finding that protamine induced TNF release by cardiomyocytes can shed new light on the understanding of protamine cardiotoxicity.

l-Arginine improves endothelial function, independently of arginine uptake, in aortas from chronic renal failure female rats

Endothelial cell dysfunction (ECD) is a common feature of chronic renal failure (CRF). Defective nitric oxide (NO) generation due to decreased endothelial nitric oxide synthase (eNOS) activity is a crucial parameter characterizing ECD. Decreased activity of cationic amino acid transporter-1 (CAT-1), the selective arginine transporter of eNOS, has been shown to inhibit eNOS in uremia. Recently, we failed to demonstrate a decrease in glomerular arginine transport in uremic female rats (Schwartz IF, Grupper A, Soetendorp H, Hillel O, Laron I, Chernichovski T, Ingbir M, Shtabski A, Weinstein T, Chernin G, Shashar M, Hershkoviz R, Schwartz D. Am J Physiol Renal Physiol 303: F396-F404, 2012). The current experiments were designed to determine whether sexual dimorphism which characterizes glomerular arginine transport system in uremia involves the systemic vasculature as well and to assess the effect of L-arginine in such conditions. Contractile and vasodilatory responses, ultrastructural changes, and measures of the L-arginine-NO system were performed in thoracic aortas of female rats subjected to 5/6 nephrectomy. The contractile response to KCl was significantly reduced, and acetylcholine-induced vasodilation was significantly impaired in aortas from CRF dames compared with healthy rats. Both of these findings were prevented by the administration of arginine in the drinking water. The decrease in both cGMP generation, a measure of eNOS activity, and aortic eNOS and phosphorylated eNOS abundance observed in CRF rats was completely abolished by l-arginine, while arginine transport and CAT-1 protein were unchanged in all experimental groups. Arginine decreased both serum levels of advanced glycation end products and the asymmetrical dimethylarginine/arginine ratio and restored the endothelial ultrastructure in CRF rats. In conclusion. arginine administration has a profound beneficial effect on ECD, independently of cellular arginine uptake, in CRF female rats.

Comparison of vasoactive response of left and right internal thoracic arteries to isosorbide-dinitrate and nitroglycerin: an in vitro study.

Abstractu2003 Objective: The internal thoracic artery (ITA) is the most important graft in coronary artery bypass grafting. Its distal region is, however, prone to vasospasm. We studied the effects of nitroglycerin (NTG) and isosorbide‐dinitrate (DSDN) on distal segments of left versus right ITA. Methods: Rings of distal segments (6 to 9 mm proximal to bifurcation) of the human left and right ITA were studied. After baseline contraction of the rings, achieved using 60 mmol/L of KCl, they were exposed to increasing doses of ISDN and NTG (10 to 100 μg/ml), and dose‐response curves were recorded. Results: The contractile response of left ITA rings to KCl were significantly lower than those of right ITA rings (1.87 ± 0.25 g versus 3.5 ± 0.61 g, p < 0.005). Both nitrates inhibited the contractile response in a concentration‐dependent manner, with relaxing effects of ISDN higher than those of NTG (p < 0.01) in both left and right ITA rings. Conclusions: The distal segment of the left ITA is less prone to vasospasm than that of the right. ISDN has a considerably higher relaxant effect on this segment than NTG. We therefore recommend favoring high doses of ISDN over NTG as an antispastic measure. (J Card Surg 2003; 18:279‐285)