Simone Tomasi

Abstract: A single-site mutant (M5) of native urokinase plasminogen activator (prouPA) induces effective thrombolysis in dogs with venous or arterial thrombosis with a reduction in bleeding complications compared to tPA. This effect, related to inhibition of two-chain M5 (tcM5) by plasma C1-inhibitor (C1I), thereby preventing non-specific plasmin generation, was augmented by the addition of exogenous C1I to plasma in vitro. In the present study, tPA, M5 or placebo +/- C1I were administered in two rat stroke models. In Part-I, permanent MCA occlusion was used to evaluate intracranial hemorrhage (ICH) by the thrombolytic regimens. In Part II, thromboembolic occlusion was used with thrombolysis administered 2 h later. Infarct and edema volumes, and ICH were determined at 24 h, and neuroscore pre (2 h) and post (24 h) treatment. In Part I, fatal ICH occurred in 57% of tPA and 75% of M5 rats. Adjunctive C1I reduced this to 25% and 17% respectively. Similarly, semiquantitation of ICH by neuropathological examination showed significantly less ICH in rats given adjunctive C1I compared with tPA or M5 alone. In Part-II, tPA, M5, and M5+C1I induced comparable ischemic volume reductions (>55%) compared with the saline or C1I controls, indicating the three treatments had a similar fibrinolytic effect. ICH was seen in 40% of tPA and 50% of M5 rats, with 1 death in the latter. Only 17% of the M5+C1I rats showed ICH, and the bleeding score in this group was significantly less than that in either the tPA or M5 group. The M5+C1I group had the best Benefit Index, calculated by dividing percent brain salvaged by the ICH visual score in each group. In conclusion, adjunctive C1I inhibited bleeding by M5, induced significant neuroscore improvement and had the best Benefit Index. The C1I did not compromise fibrinolysis by M5 in contrast with tPA, consistent with previous in vitro findings.

Abstract: M5 is a mutant of the fibrin-specific proenzyme, single-chain urokinase-type plasminogen activator. M5 has the unusual property that its non-specific two-chain enzymatic form (tcM5) is inhibited rapidly by plasma C1-inhibitor (C1I). As a result, non-specific plasminogen activation, responsible for the hemorrhagic complications of plasminogen activators, can be prevented by C1I. Exceptionally, the rat endogenous C1I does not significantly inhibit tcM5, making rats unusually sensitive both to the side effects of M5 and to the salutary effects of added C1I. Two stroke models were used in a proof of concept study: (I) irreversible ischemia by ligation of the ICA and cauterization of the MCA in order to evaluate ICH. Thrombolytic infusions (30 min) were administered 4h later ; (II) reversible ischemia by thromboembolic occlusion of the MCA and infusions 2h later. Rats were given either tPA (10mg/Kg) or M5 (15mg/Kg) ?/- an adjunctive bolus of C1I or saline ?/- C1I. In model I, ICH mortality was high with both tPA (57%) and M5 (75%) but was reduced significantly by C1I to 25% and 17% respectively. In model II, M5?C1I induced the lowest infarct volume and caused the least non-lethal ICH (17%) and was the only group among the six to achieve significant reduction in functional neuroscore between pretreatment (3.30.2) and 24h later (2.00.4). The infarct volumes of tPA alone and M5?C1 were comparable (9221 and 8815 mm3 respectively), indicating a comparable fibrinolytic effect. However, tPA alone caused much more ICH and was accompanied by the greatest edema volume (64mm3 or 70% of the infarct volume), consistent with tPA’s known disruption of the blood brain barrier. Adjunctive C1I increased the tPA infarct volume to 14215 mm3, making it no longer significantly less than controls (21328 and 22713). However, C1I also reduced the tPA edema volume significantly to 24% of infarct volume. Activation of the complement pathway by tPA has been previously described by others, which may explain this C1I effect. In conclusion, the combination M5?C1I was as effective as tPA alone and more effective than tPA?C1I, causing significantly less ICH, edema volume, and the most functional improvement. These in vivo findings with M5 validate the in vitro data with C1I and provide a promising development for a new, effective and safer treatment of ischemic stroke.

Abstract: The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The d-retro-inverso form of c-Jun N-terminal kinase-inhibitor (D-JNKI1), a cell-permeable inhibitor of JNK, powerfully reduces neuronal death induced by permanent and transient ischemia, even when administered 6 h after the ischemic insult, offering a clinically relevant window. We investigated the JNK molecular cascade activation in rat cerebral ischemia and the effects of D-JNKI1 on this cascade. c-Jun activation starts after 3 h after ischemia and peaks at 6 h in the ischemic core and in the penumbra at 1 h and at 6 h respectively. The 6 h c-Jun activation peak correlates well with that of P-JNK. We also examined the activation of the two direct JNK activators, MAP kinase kinase 4 (MKK4) and MAP kinase kinase 7 (MKK7). MKK4 showed the same time course as JNK in both core and penumbra, reaching peak activation at 6 h. MKK7 did not show any significant increase of phosphorylation in either core or penumbra. D-JNKI1 markedly prevented the increase of P-c-Jun in both core and penumbra and powerfully inhibited caspase-3 activation in the core. These results confirm that targeting the JNK cascade using the TAT cell-penetrating peptide offers a promising therapeutic approach for ischemia, raising hopes for human neuroprotection, and elucidates the molecular pathways leading to and following JNK activation.

Abstract: Beta-nicotinamidedinucleotide phosphate diaphorase (NADPH-d) colocalizes with NOS in the central nervous system. Two types of NADPH-d-positive neurons are present in the primate cerebral cortex: type 1, intensely and Golgi-like labeled neurons, a subset of GABAergic interneurons; type 2, lightly labeled neurons (divided into two subclasses, a first one having a lightly stained cell body bearing only one short process, and a second one showing intense NADPH-d staining with short processes extending radially). We have analyzed the distribution of NADPH-d activity in human frontal, temporal, and occipital cortical areas, finding remarkable laminar and interareal differences in cell size and distribution of the different cell types. There was a clear bias for type 1 neurons in infragranular layers in all areas considered; both in supra- and infragranular layers, their density was highest in frontal, and lowest in temporal cortex. The density of type 2 neurons was lower supragranularly in temporal cortex and infragranularly in occipital cortex. The overall density of type 2 cells was remarkably higher in occipital cortex than in the temporal and frontal ones. Type 1 neurons were significantly larger than type 2, and were smaller in the supragranular than in the infragranular subzone in occipital and temporal cortex. Type 1 cells were significantly larger in frontal cortex than in occipital and temporal cortex, and type 2 cells were significantly smaller in occipital than in temporal and frontal cortex. These area-related differences might reflect differences between heterotypic and homotypic cortex in the regulation of cortical blood flow.