Apoptotic hepatocytes have been demonstrated to represent an important signal for transmigration of leukocytes sequestered in sinusoids during endotoxemia in vivo. Beside leukocytes, platelets and their adhesion to endothelial cells and leukocytes have been implicated in inflammatory liver injury. Using in vivo multifluorescence microscopy, we examined the possibility that hepatocellular apoptosis causes both leukocytes and platelets to colocalize within the sinusoidal microvasculature of endotoxemic livers. We further addressed the issue whether cellular colocalization with apoptotic hepatocytes is cause or consequence of apoptosis. Intraperitoneal exposure of rats with LPS (5 mg/kg) induced liver injury after 6 and 16 h, as given by nutritive perfusion failure (20 +/- 2 and 21 +/- 2%), intrahepatic leukocyte (60 +/- 10 and 121 +/- 48 cells/mm(2)), and platelet (12 +/- 4 and 34 +/- 4 cells/mm(2)) accumulation as well as parenchymal cell apoptosis (4 +/- 1 and 11 +/- 2 cells/mm(2)) and caspase cleavage (4.7 +/- 2.4- and 7.0 +/- 3.0-fold increase; P < 0.05 vs. saline-exposed controls). Higher doses of LPS (10 mg/kg ip) further increased intrahepatic leukocyte and platelet accumulation but not the extent of parenchymal apoptosis. Detailed spatial analysis revealed colocalization of leukocytes (range 12-24%) but barely of platelets (<6%) with apoptotic hepatocytes in all endotoxemic groups studied. It is of interest, however, that platelets were found at increasing rates in colocalization with leukocytes at 6 and 16 h after LPS exposure (5 mg/kg LPS: 7 +/- 3 and 25 +/- 6%; 10 mg/kg LPS: 11 +/- 4 and 14 +/- 1%). Platelet-leukocyte events significantly correlated with the extent of caspase cleavage as an indicator of tissue apoptosis (P < 0.05; r = 0.82). Blockade of apoptosis by a pan-caspase inhibitor caused a significant reduction of leukocyte adherence and platelet-leukocyte colocalization on LPS exposure. On the other hand, leukocytopenic animals revealed reduced hepatocyte apoptosis, although values still exceeded those of controls, and in leuko- and thrombocytopenic animals, hepatocyte apoptosis was found reduced to control values. Taken together, LPS-associated hepatocyte apoptosis seems to be initiated by circulating blood cells that become adherent within the liver but might also contribute to further sustain the inflammatory cell-cell response.

Objective-By heme degradation, heme oxygenase-1 (HO-1) provides endogenous carbon monoxide and bilirubin, both of which play major roles in vascular biology. The current study aimed to examine whether induction of HO-1 and its byproducts modulate the process of microvascular thrombus formation in vivo. Methods and Results-In individual microvessels of mouse cremaster muscle preparations, ferric chloride-induced thrombus formation was analyzed using intravital fluorescence microscopy. When mice were pretreated with an intraperitoneal injection of hemin, a HO-1 inducer, immunohistochemistry and Western blot protein analysis of cremaster muscle tissue displayed a marked induction of HO-1. In these animals, superfusion with ferric chloride solution induced arteriolar and venular thrombus formation, which, however, was significantly delayed when compared with thrombus formation in animals without HO-1 induction. The delay in thrombus formation in hemin-treated mice was completely blunted by tin protoporphyrin-IX, a HO-1 inhibitor, but not by copper protoporphyrin-IX, which does not inhibit the enzyme. Coadministration of the vitamin E analogue Trolox in HO-1-blocked animals almost completely restored the delay in thrombus formation, implying that, besides CO, the antioxidant HO pathway metabolite bilirubin mainly contributes to the antithrombotic property of HO-1. This was further supported by the fact that bilirubin was found as effective as hemin in delay of ferric chloride-induced thrombus formation. Animals with HO-1 induction revealed reduced P-selectin protein expression in cremaster muscle tissue, which most probably presented the molecular basis for delayed thrombus growth. Conclusion-Local

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Nicotine as one toxic component of cigarette smoke does not affect vascularization, but adversely influences follicular growth by an increase in apoptotic cell death. As follicular growth is a crucial step in normal ovulation and fertilization, nicotine-induced cell apoptosis may represent one of the mechanisms underlying the well-established link between smoking and fertility disorders.

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Abstract-In aged spontaneously hypertensive rats (SHR), vasorelaxant responses to NO are attenuated compared with normotensive control rats (Wistar-Kyoto [WKY]) because of a decreased expression of the important NO receptor soluble guanylyl cyclase (sGC). Because the expression of sGC subunits ␣ 1 and ␤ 1 is controlled at the posttranscriptional level by the mRNA-binding protein human-antigen R (HuR), we now assessed whether or not altered expression of HuR could account for downregulation of sGC␣ 1 and sGC␤ 1 in genetic hypertension. The expression of HuR (and sGC␣ 1 and sGC␤ 1 ) in aortas from aged SHR was significantly decreased at the mRNA and protein level compared with age-matched WKY rats, whereas expression of HuR was not different in prehypertensive young (2 months of age) SHR and age-matched WKY rats. The mRNA-binding activity of HuR in native aortic protein extracts from aged SHR was markedly reduced compared with normotensive WKY rats, as detected by RNA electrophoretic mobility shift analysis, using biotin-labeled adenine and uracil (AU)-rich element (ARE)-containing RNA probes from the 3Ј-untranslated region of sGC␣ 1 and sGC␤ 1 . In contrast, ARE-binding activity was not different between prehypertensive young SHR and young WKY rats. In vitro RNA degradation assays using the same AU-rich sGC mRNA probes revealed an accelerated sGC␣ 1 and sGC␤ 1 mRNA decay in the presence of native protein extract from hypertensive SHR, which was less rapid with aortic protein from normotensive WKY rats. These findings suggest that in this animal model of genetic hypertension, the reduced expression of sGC subunits is mediated by downregulation of the sGC mRNA-stabilizing protein HuR. Key Words: rats, spontaneously hypertensive Ⅲ aorta Ⅲ gene regulation Ⅲ hypertension, genetic C hronic hypertension is associated with functional and morphological alterations of the vessel wall (ie, dysfunctional vascular endothelium and thickening of the smooth muscle layer). The pathomechanisms accounting for hypertension-induced vascular alterations are likely to be multifactorial. A major homeostatic factor in the vessel wall is NO, which is generated from L-arginine by endothelial NO synthase. NO reduces vascular tone by activation of soluble guanylyl cyclase (sGC) and stimulation of cGMP formation and cGMP-activated protein kinase-I (cGK-I). Activated cGK-I reduces vascular tone by interference with intracellular Ca 2ϩ mobilization 1 and inhibition of contractile filament function. 2 In animal models of hypertension such as the spontaneously hypertensive rat (SHR), disturbances of the NO-cGMP system contribute to vascular dysfunction. Thus, increased oxidative stress interfering with endogenous NO bioavailability and reduced expression of sGC interfering with NO downstream signaling have been reported. 3-5 sGC is a heterodimeric hemoprotein consisting of ␣ 1 (76 to 81.5 kDa) and ␤ 1 (70 kDa) subunits. 6 We have shown recently that the expression of sGC is subject to post-transcriptional regulation. 7 The elav-like (embryonic-le...

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In order to study protein degradation during flight in homing, a high-performance liquid chromatography technique was developed for the quantitative analysis of N tau-methylhistidine. Secondly, it was necessary to confirm that the excretion of N tau-methylhistidine correlates with myofilament breakdown in homing pigeons. In these experiments, ten birds were subcutaneously injected with N tau-[14C]methylhistidine and the excreta were quantitatively collected for 1 week. Of the 94.5% radioactivity recovered, 87.1% was associated with N tau-[14C]methylhistidine and 6.1% with N-acetyl-N tau-[14C]methylhistidine. This rapid excretion of unmetabolized N tau-[14C]methylhistidine validates the assumption that the amount of N tau-methylhistidine excreted is a measure of myofilament catabolism in homing pigeons. The influence of endurance flight on protein breakdown was determined after flights from release sites 368-646 km away. Immediately after return, plasma urea and uric acid levels were increased, whereas plasma concentration of N tau-methylhistidine remained unchanged compared to unflown control birds. Flown pigeons excreted significantly more urea and N tau-methylhistidine within 24 h and significantly more urea and uric acid within 96 h after flight than unflown controls. Our findings support the hypothesis that in homing pigeons protein catabolism is increased during endurance flight. Elevated N tau-methylhistidine excretion probably results from repair processes in damaged muscle fibers, including breakdown of myofilaments.