J. Nanobashvili

l-Arginine Treatment Alters the Kinetics of Nitric Oxide and Superoxide Release and Reduces Ischemia/Reperfusion Injury in Skeletal Muscle

BACKGROUND Constitutive nitric oxide synthase (cNOS) may produce species involved in ischemia/reperfusion (I/R) injury: NO in the presence of sufficient L-arginine and superoxide at the diminished local L-arginine concentration accompanying I/R. METHODS AND RESULTS During hindlimb I/R (2.5 hours/2 hours), in vivo NO was continuously monitored (porphyrinic sensor), and L-arginine (chromatography), superoxide (chemiluminescence), and I/R injury were measured intermittently. Normal rabbits were compared with those infused with L-arginine 4 mg x kg(-1) x min(-1) for 1 hour. In both groups, approximately 6 minutes into ischemia, a rapid increase of NO from its basal level of 50+/-17 to 115+/-7 nmol/L, P<.005 (microvessels), was observed. In animals not treated with L-arginine, NO dropped below basal to undetectable levels (<1 nmol/L) during reperfusion. In animals treated with L-arginine, the decrease of NO was slower, such that substantial amounts accumulated during reperfusion (25 nmol/L). Decreased NO during I/R was accompanied by increased superoxide, which during reperfusion reached 50 nmol/L without or 23 nmol/L with L-arginine treatment. Calcium-dependent cNOS was a major source of superoxide release (inhibited 70% by L-NMMA and 25% by L-NAME) during I/R. CONCLUSIONS L-Arginine treatment decreased superoxide generation by cNOS while increasing NO accumulation, leading to protection from constriction (microvessel area, 17.77+/-0.95 versus 11.66+/-2.21 microm2 untreated, P<.0005) and reduction of edema after reperfusion (interfiber area, 16.56+/-2.13% versus 27.68+/-7.70% untreated, P<.005).

S-Nitroso Human Serum Albumin Treatment Reduces Ischemia/Reperfusion Injury in Skeletal Muscle via Nitric Oxide Release

Background—Peroxynitrite generated from nitric oxide (NO) and superoxide (O2−) contributes to ischemia/reperfusion (I/R) injury. Feedback inhibition of endothelial NO synthase by NO may inhibit O2− production generated also by endothelial NO synthase at diminished local l-arginine concentrations accompanying I/R. Methods and Results—During hindlimb I/R (2.5 hours/2 hours), in vivo NO was monitored continuously (porphyrinic sensor), and high-energy phosphates, reduced and oxidized glutathione (chromatography), and I/R injury were measured intermittently. Rabbits receiving human serum albumin (HSA) (controls) were compared with those receiving S-nitroso human serum albumin (S-NO-HSA) beginning 30 minutes before reperfusion for 1 hour or 30 minutes before ischemia for 3.5 hours (0.1 &mgr;mol · kg−1 · h− 1). The onset of ischemia led to a rapid increase of NO from its basal level (50±12 nmol/L) to 120±20 and 220±15 nmol/L in the control and S-NO-HSA–treated groups, respectively. In control animals, NO dropped below basal levels at the end of ischemia and to undetectable levels (<1 nmol/L) during reperfusion. In S-NO-HSA–treated animals, maximal NO levels never decreased below basal concentration and on reperfusion were 100±15 nmol/L (S-NO-HSA preischemia group, 175±15 nmol/L). NO supplementation by S-NO-HSA led to partial and in the preischemia group to total preservation of high-energy phosphates and glutathione status in reperfused muscle (eg, preischemia groups: ATP, 30.23±5.02 &mgr;mol/g versus control, 15.75±4.33 &mgr;mol/g, P <0.0005; % oxidized glutathione, 4.49± 1.87% versus control, 22.84±6.39%, P <0.0001). S-NO-HSA treatment in all groups led to protection from vasoconstriction and reduced edema formation after reperfusion (eg, preischemia groups: interfiber area, 12.94±1.36% versus control, 27.83±1.95%, P < 0.00001). Conclusions—Long-lasting release of NO by S-NO-HSA provides significant protection of skeletal muscle from I/R injury.

Prostaglandin E1 reduces ischemia/reperfusion injury by normalizing nitric oxide and superoxide release.

To test the effects of prostaglandin E1 on 2.5 h of ischemia followed by 2 h of reperfusion, continuous nitric oxide measurements (electrochemical) were correlated with intermittent assays of superoxide and peroxynitrite levels (chemiluminescence) and ischemia/reperfusion injury in rabbit adductor magnus muscle. Administering prostaglandin E1 (1 microg/kg) before or during ischemia/reperfusion caused normalization of the release of nitric oxide, superoxide, and peroxynitrite to slightly above preischemic levels. This pattern was dramatically different from that observed during ischemia/reperfusion alone, where nitric oxide concentration increased three times above its basal level. Normalization of constitutive nitric oxide synthase activity in the presence of prostaglandin E1 was associated with a significant reduction of superoxide and peroxynitrite production and subsequent reduction of ischemia/reperfusion injury. At 2 h of reperfusion, vasoconstriction associated with ischemia/reperfusion injury was eliminated, and edema was significantly mollified but still apparent. Prostaglandin E1 treatment does not directly inhibit constitutive nitric oxide synthase, like the inhibitor N(omega)-monomethyl-L-arginine. Some phenomenon associated with ischemia turns on endothelial constitutive nitric oxide synthase to start transforming L-arginine and oxygen into nitric oxide, but prostaglandin E1 seems to inhibit this phenomenon. Thus, essential local L-arginine pools are not depleted, and normal basal levels of essential nitric oxide are maintained, whereas cytotoxic superoxide and peroxynitrite production by L-arginine-deficient constitutive nitric oxide synthase is prevented.

Effect of α-tocopherol pretreatment on high energy metabolites in rabbit skeletal muscle after ischemiareperfusion

The ability of skeletal muscle to recover high energy phosphate compounds in response to pretreatment with vitamin E was investigated in a rabbit hindlimb ischemia/reperfusion model (2.5 h/2 h). High energy metabolites were measured in the adductor magnus muscle of untreated animals and compared to the treatment group (all rac-α-tocopheryl acetate, 3 mg/kg body weight, supplemented i.v. before the onset of ischemia). Phosphocreatine (PCr) levels decreased after ischemia more than 65% in untreated and treatment groups, but tended to recover in treatment group after reperfusion. Adenosine triphosphate (ATP) values decreased by 50% of basal level after reperfusion in the untreated group, whereas α-tocopherol pretreatment prevented ATP depletion.

Multivitamin administration before ischemia reducesischemia-reperfusion injury in rabbit skeletal muscle

This study investigated the effect of a multivitamin preparation administered before ischemia or before reperfusion, on ischemia-reperfusion (I/R) injury of skeletal muscle. An in vivo hindlimb skeletal muscle I/R model (2.5 h/2 h) was carried out on adult New Zealand white rabbits. Animals used as I/R models were treated with a multivitamin preparation (0.4 ml/kg bw i.v. bolus), containing alpha-tocopherol, ascorbic acid, retinol, vitamin B complex, 30 min before starting ischemia (group MV(isc)) or 5 min before reperfusion (group MV(rep)) and compared to animals with I/R without treatment (group IR) and sham operated animals (group SHAM). Interstitial edema (muscle interfiber area, %MIFA) and changes in microvessel size (microvessel cross sectional area, MVCSA, microm(2)) were measured. Plasma malondialdehyde concentrations (MDA-TBA, nmol/ml) served as a measure of lipid peroxidation. After 2h of reperfusion, ischemia-reperfusion developed a significant microvascular constriction and an interstitial edema (IR, vs SHAM;P<< 0.01), but administration of antioxidative vitamins before the onset of ischemia reduced microvascular constriction and edema formation (P<< 0.05 vs IR group). In a similar manner, administration of vitamins before ischemia lowered plasma MDA-TBA levels as compared to the untreated group during reperfusion (p<< 0. 05). In animals treated with vitamins before reperfusion, the biochemical and morphological results showed no differences as compared to the untreated group. Antioxidative treatment with a multivitamin preparation exerted a beneficial effect on I/R injury of skeletal muscle when the aforementioned vitamins were administered before ischemia but not before the onset of reperfusion.

Influence of a short-term acute ischemia and reperfusion on skeletal muscle metabolism and morphology in rabbits

The duration of the ischemia and reperfusion of the limbs determines the extent of tissue damage. The purpose of this study was to validate anin vivo model of ischemia-reperfusion injury with a short-term ischemic period of 1 hour which is comparable to several clinical situations. Thirteen New Zealand rabbits were randomly assigned to either (1) the sham operation (control), or (2) ischemiareperfusion group response to 1 hour of ischemia followed by 2 hours of reperfusion. Ischemia was created by clamping bilateral common femoral arteries and tourniquet occlusion of collateral circulation. Alterations of plasma potassium, malondialdehyde (MDA), muscle tissue intercellular thromboxane B2 (TxB2) concentrations, and morphometric determinations of skeletal muscle were parameters used to quantify reperfusion injury. Interstitial edema (median fraction of muscle interfiber area was 16.57% vs 13.39% in control,p=0.02), muscle cell shrinkage (median muscle fiber area was 3.70×103 μm2 vs 4.55×103 in control,p= 0.04), increased plasma potassium (median 4.7 mmol/L vs 3.6,p=0.02) in 2 hours and increased interstitial TxB2 in 1 hour were manifestation of reperfusion injury. We conclude that expansion of the extracellular space not accompanied by changes of plasma MDA may indicate that lipidperoxidation in reperfused muscle was negligible and interstitial edema was related to thromboxane A2 synthesis.

Gutless Adenoviral Vectors – Promising Tools for Gene Therapy

SummaryBackground: Progress in gene therapy depends on establishing the appropriate gene transfer methods. The most efficient vehicles for the delivery of foreign genes to the target tissues are modified adenoviruses. Methods: Adenoviral vectors of the first generation despite the high infection efficiency, have an essential drawback: They induce a strong immune response, which excludes them from clinical trials. Results: In the past few years, there have been substantial efforts to improve adenoviral vectors with particular emphasis on reducing immunological effects in the host organism. Conclusions: In this review we describe the preparation and application of gutless vectors, the most advanced and promising type of adenoviral vehicles so far.ZusammenfassungGrundlagen: Der Erfolg einer Gentherapie hängt von der Etablierung geeigneter Gentransfermethoden ab. Die effektivsten Transportmittel für Fremdgene zum Zielgewebe vermitteln modifizierte Adenoviren. Methodik: Die erste Generation von adenoviralen Vektoren haben ungeachtet der hohen Infektiosität einen essentiellen Nachteil: sie induzieren eine starke Immunantwort, die sie von klinischen Versuchen ausschließt. Ergebnisse: In den letzten Jahren gab es verstärkte Bemühungen, die adenoviralen Vektoren zu verbessern und im Besonderen die immunologischen Effekte an dem Empfängerorganismus zu reduzieren. Schlußfolgerungen: Hier beschreiben wir die Präparation und Applikation von „Gutless“-Vektoren, den bisher bestentwickelten und vielversprechendsten adenoviralen Vektoren.

α‐Tocopherol Pretreatment Reduces Ischaemia/Reperfusion Injury in Rabbit Skeletal Muscle

SummaryBackground: Ischaemia/reperfusion (I/R) injury is characterized by the production of oxygen free radicals and the loss of high energy phosphates. α-Tocopherol (vitamin E) has been reported to play a protective role against free radical damage. Therefore, its i. v. supplementation prior to onset of ischaemia was investigated in an established I/R injury model.Methods: New Zealand white rabbits were subjected to a bilateral hind limb ischaemia/reperfusion of 2.5 h/2 h without any treatment (IR group), whereas in the VE group the vitamin E emulsion Tocovenös® was given i. v. for 10 min in a dose of 3 mg/kg bw. A SHAM group served as a control. Intramuscular phosphocreatine (PCr) and adenosine triphosphate (ATP), muscle interfibre area (MIFA), and microvessel cross-sectional area (MVCSA) were determined and electron microscopy was performed.Results: In the IR group, PCr decreased by 65 % of the initial level after 2.5 h of ischaemia and a further depletion was noticed after 2 h of reperfusion (20.9±1.1 vs 6.3±3.0 µmol/g wet wt,P<0.01). By contrast, ATP remained stable after ischaemia but dropped dramatically after reperfusion (6.5±0.2 vs 2.1±8.0 µmol/g wet wt,P<0.05). In the VE group, PCr and ATP did not differ significantly from the SHAM group at the end of reperfusion. Consequently, ATP in this group was significantly higher as compared with the IR group (4.9±0.9 vs 2.1±8.0 µmol/g wet wt,P<0.05). In the IR group, prominent interstitial oedema formation was found after reperfusion expressed by the increase in MIFA (28.2±4.2 vs 13.7±1.6 %,P<0.05). In the VE group, only a moderate interstitial oedema was found (18.7±3.4 %). Severe microvessel constriction was observed after reperfusion in both experimental groups. Electron microscopy revealed fewer tissue changes in the VE group as compared with the IR group.Conclusions: α-Tocopherol pretreatment had a protective effect on I/R injury in skeletal muscle expressed by recovery of PCr after reperfusion and by prevention of ATP depletion. Although microvascular constriction could not be prevented, antioxidative treatment reduced interstitial oedema and preserved muscle morphology.ZusammenfassungGrundlagen: Der Ischämie/Reperfusions (I/R)-Schaden ist durch die Produktion freier Sauerstoffradikale und den Verlust energiereicher Phosphate charakterisiert. Es ist bekannt, daß α-Tocopherol (Vitamin E) eine Schutzfunktion bei Schäden durch freie Radikale hat. Deshalb wurde eine i. v. Supplementation vor Beginn der Ischämie an einem etablierten I/R-Modell untersucht.Methodik: An weißen New Zealand Kaninchen wurde eine beidseitige Ischämie/Reperfusion von 2,5 h/2 h an den hinteren Extremitäten durchgeführt: in Gruppe I/R ohne Behandlung. In der VE-Gruppe wurde die Vitamin-E-Emulsion Tocovenös® i. v. für 10 min in einer Dosierung von 3 mg/kg gegeben. Eine SHAM-Gruppe diente als Kontrolle. Intramuskuläres Phosphokreatin (PCr), Adenosin Triphosphat (ATP), die Fläche zwischen den Muskelfasern (MIFA) und die Mikrogefäßfläche (MVCSA) wurden bestimmt und elektronenmikroskopische Aufnahmen ausgewertet.Ergebnisse: In der IR-Gruppe verringerte sich das PCr nach 2,5 h Ischämie um 65 % im Verhältnis zum Ausgangswert. Nach 2 h Reperfusion wurde eine weitere Absenkung gemessen (20,9±1,1 vs 6,3±3,0 µmol/g wet wt,P<0,01). Im Gegensatz dazu blieb der ATP-Spiegel während der Ischämie stabil, fiel aber stark nach der Reperfusion (6,5±0,2 vs 2,1±8,0 µmol/g wet wt,P<0,05). In der VE-Gruppe veränderten sich PCr und ATP nicht signifikant zur SHAM-Gruppe am Ende der Reperfusion und ATP war im Vergleich zur IR-Gruppe signifikant höher (4,9±0,9 vs 2,1±8,0 µmol/g wet wt,P<0,05). In der IR-Gruppe zeigte sich am Ende der Reperfusion ein deutliches interstitielles Ödem (Anstieg der MIFA) im Vergleich zur SHAM-Gruppe (28,2±4,2 vs 13,7±1,6 %,P<0,05). In der VE-Gruppe wurde nur ein geringes interstitielles Ödem gemessen (18,7±3,4 %). In beiden experimentellen Gruppen wurde eine deutliche Konstriktion der Mikrogefäße beobachtet. Die Elektronenmikroskopie zeigte in der VE Gruppe geringere Gewebsveränderungen als in der IR-Gruppe.Schlußfolgerungen: Eine α-Tocopherol-Vorbehandlung hat einen protektiven Effekt auf die Skelettmuskulatur beim I/R-Schaden, erkennbar an der Normalisierung des PCr nach Reperfusion und der Vorbeugung eines ATP-Abfalls. Obwohl eine Konstriktion der Mikrogefäße nicht verhindert werden konnte, reduzierte die antioxidative Behandlung das interstitielle Ödem und bewahrte die Muskelmorphologie.

Vasculogenesis - a new strategy for induction of peripheral neovascularization

SummaryBackground: New blood vessel development can be induced by supplementation with angiogenic growth factors. In some older diabetic or hypercholesterolaemic patients, however, endothelial cells are defective and cannot support angiogenesis. Methods: Endothelial progenitor cells (EPC) can help to overcome this limitation, providing a source of viable endothelial cells. Results: It has been demonstrated that EPC can improve neovascularization in ischaemic hind limb, accelerate blood flow in diabetic mice, and improve cardiac function. Conclusions: In this review, we will summarize the effects of EPC in the treatment for experimental peripheral ischaemia.ZusammenfassungGrundlagen: Die Entwicklung neuer Blutgefäße kann durch die Gabe von angiogenetischen Wachstumsfaktoren induziert werden. Bei älteren Zuckerkranken oder Hypercholesterinämie-Patienten sind die Endothelzellen geschädigt und können die Angiogenese nicht unterstützen. Methodik: Mit Hilfe der endothelialen Progenitorzellen (EPC) können funktionsfähige Endothelzellen gebildet werden. Ergebnisse: Es konnte gezeigt werden, daß die EPC zu einer Neovaskularisation in ischämischen Extremitäten führt, den Blutfluß bei diabetischen Mäusen und die Herzfunktion verbessert. Schlußfolgerungen: In diesem Review diskutieren wir die Effekte der EPC bei der Behandlung experimenteller peripherer Ischämien.

Gefäßzentren aus chirurgischer Sicht: Gefäßmedizin an der Universitätsklinik Wien / Centers of Vascular Medicine at the University Clinic in Vienna

Die Entwicklung von der klassisch-mechanistischen Gefaschirurgie hin zu einer interdisziplinaren Gefasmedizin wird am Beispiel der Univ.-Klinik Wien dargestellt. Nach Grundung eines Ludwig-Boltzmann-Institutes fur interdisziplinare klinische Gefasmedizin 1998 ist die Anzahl der Vortrage der Mitarbeiter der Klin. Abt. f. Gefaschirurgie von 106 auf 188, und der Impact-Factor der Publikationen von 27,7 auf 119,0 angestiegen (Vergleich: Zeitraum 1994 bis 1996 mit 1997 bis 1999). Der Gesamt-Impact-Factor der Publikationen der Abteilungen fur Gefaschirurgie, Angiologie und Interventionelle Radiologie konnte von 147,0 auf 291,3 gesteigert werden. Die Drittmittelfinanzierung in der autonomen Rechtsfahigkeit konnte im Jahr 1999 um mehr als 60% im Vergleich zu 1996 erhoht werden.