Nicotine does not affect vascularization but inhibits growth of freely transplanted ovarian follicles by inducing granulosa cell apoptosis.

BACKGROUND: There is clear support for an association between smoking and decreased female fecundity and fertility. Cigarette smoke appears to have adverse effects along a continuum of reproductive processes. We therefore studied the effect of nicotine on follicular growth and vascularization of freely transplanted ovarian follicles. METHODS: We used the skinfold chamber model in Syrian golden hamsters, which allows the in vivo microscopy of follicular grafts. Animals were treated daily with nicotine at doses mimicking low-rate and high-rate smokers (0.2 and 1.0 mg/kg body weight subcutaneously). Saline-treated animals served as controls. To further evaluate the effect of nicotine on angiogenesis, an in vitro aortic ring assay was used. RESULTS: The re-vascularisation rate of follicles was similar in nicotine-treated animals and controls. During the 7 days after transplantation, nicotine further caused a dose-dependent inhibition of follicular growth. In contrast, the vascularized area and microvessel density were not affected by the nicotine exposure. In vitro aortic ring assays confirmed that nicotine does not influence sprouting and microvessel formation. However, immunohistochemistry for cleaved caspase-3 revealed a large extent of granulosa cell apoptosis within transplanted follicles of high-dose nicotine-treated animals. CONCLUSIONS: 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.

Inhibition of p53 during physiological angiogenesis in the hamster ovary does not affect extent of new vessel formation but delays vessel maturation.

Transcription factor p53 regulates the cell cycle and apoptosis and may impair angiogenesis by the deregulation of pro-angiogenic factors and the activation of anti-angiogenic factors. Our aim has been to elucidate further the role of p53 in physiological angiogenesis. By treating hamsters with the wildtype p53 inhibitor pifithrin-alpha (PFT) versus equivalent volumes of the vehicle dimethylsulfoxide, we showed a reduced p53 tissue protein level, a reduction of poly(ADP-ribose) polymerase and cleaved caspase-3 products, and a slightly increased proliferation of cell nuclear antigen and cyclin D1 by Western blot protein analysis of ovarian tissue. PFT further increased platelet-derived growth factor and did not influence vascular endothelial growth factor in female reproductive tissue. Despite these differences in tissue levels of proteins potentially involved in angiogenesis, in vivo fluorescence-microscopic analysis of freely transplanted ovarian follicles revealed comparable kinetics and an extent of revascularization with almost identical densities of network microvessels in both groups. However, follicles of PFT-treated animals exhibited enlarged diameters and higher volumetric blood flow within the newly formed microvessels. Less-dense basement membranes with unclear laminar structure and only a loose contact of pericytes to endothelial cells were also occasionally found, providing evidence of delayed maturation and impaired diameter control of microvessels. Thus, inhibition of wildtype p53 during physiological angiogenesis does not affect the extent of new vessel formation but may delay the maturation of newly formed microvessels.

Vascular heme oxygenase-1 induction suppresses microvascular thrombus formation in vivo.

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. CONCLUSIONS: Local induction of HO-1 activity may be of preventive and therapeutic value for clinical disorders with increased risk of thrombotic events.

Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.

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.

Influence of flight on protein catabolism, especially myofilament breakdown, in homing pigeons.

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.