Tissue plasminogen activator deficiency promotes early phase regeneration in the olfactory epithelium after bulbectomy.

BACKGROUND: Tissue type plasminogen activator (tPA) functions as a fibrinolytic factor in the blood and has unique roles in the nervous system. However, the role of tPA in the olfactory epithelium (OE) is still unclear. Generally, surgical ablation of the olfactory bulb (bulbectomy) triggers degeneration followed by regeneration of OE. In this experimental study, we investigated the role of tPA in OE regeneration. METHODS: Wild-type (WT) mice and tPA-knockout (tPA(-/-) ) mice were subjected to bulbectomy. Reverse-transcription polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical examination was done to detect tPA expression in the olfactory bulb and OE. Cellular proliferation and apoptosis was also monitored in the OE. RESULTS: Before bulbectomy, tPA was found to be expressed in the olfactory bulb and OE. OE degenerated to a similar extent in both strains between 0 and 3 days after bulbectomy. However, OE was thicker and contained more cells in tPA(-/-) mice than in WT mice at 7 days after bulbectomy. Moreover, the number of apoptotic bodies was reduced and the number of proliferating cells was increased in the OE of tPA(-/-) mice compared to WT mice, after bulbectomy. Transmission electron microscopy revealed continuous degeneration of the OE for up to 7 days after bulbectomy in WT mice. In contrast, we observed some intact olfactory vesicles and almost normal supporting cells in the OE of tPA(-/-) mice, at 7 days after bulbectomy. CONCLUSION: The current findings show that the tPA-plasmin system plays an inhibitory role in the regulation of regeneration in the OE.

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source.

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

The morphological change of supporting cells in the olfactory epithelium after bulbectomy.

Transmission electron microscopy was used to study the responses of the supporting cells of the olfactory epithelium at 1-5 days after surgical ablation of the olfactory bulb (bulbectomy). In intact olfactory epithelium, lamellar smooth endoplasmic reticulum and rod-shaped mitochondria were distinctly observed in the supporting cells. On the first day after bulbectomy, bending of the microvilli and an increase in the smooth endoplasmic reticulum were observed. Cristae of the mitochondria became obscure, and the density of the mitochondrial matrix decreased. On the second day after bulbectomy, the number of microvilli decreased, broad cytoplasmic projections that contained cytoplasmic organelles protruded into the luminal side, and the mitochondria were swollen. On the fifth day after bulbectomy, microvilli seemed to be normal and some cells had large cytoplasmic projections that protruded toward the lumen of the nasal cavity. Within the cytoplasmic projections of the supporting cells, a large lamellar and reticular-shaped smooth endoplasmic reticulum was evident. Mitochondria exhibited almost normal morphology. The current findings demonstrate that morphological changes occur in the supporting cells after bulbectomy. This new evidence hypothesizes that these changes represent events that contribute to the regeneration of the olfactory epithelium after bulbectomy.

Distribution of the androgen receptor in the diencephalon and the pituitary gland in goats: Co-localisation with corticotrophin releasing hormone, arginine vasopressin and corticotrophs.

Previously it has been shown that androgen suppresses transportation-induced increases in plasma adrenocorticotropic hormone (ACTH), possibly by suppressing the secretion of corticotrophin releasing hormone (CRH) or arginine vasopressin (AVP) from the hypothalamus, or secretion of ACTH from the pituitary gland. The aim of the present study was to examine androgen target sites in the caprine diencephalon and pituitary gland using immunohistochemical methods. The androgen receptor (AR) was expressed strongly in the bed nucleus of the stria terminalis, the medial preoptic area, the arcuate nucleus, the ventromedial hypothalamic nucleus and the suprachiasmatic nucleus in the diencephalon. Between 8% and 11% of CRH and AVP neurons in the paraventricular hypothalamic nucleus (PVN) expressed AR. In the pituitary gland, 7.1% of corticotrophs expressed AR. The results are consistent with the proposal that androgen acts directly and indirectly on CRH and/or AVP neurons in the PVN. The possibility of a direct action of androgen on the corticotrophs in the pituitary gland was also considered.

Non-cell autonomous impairment of oligodendrocyte differentiation precedes CNS degeneration in the Zitter rat: implications of macrophage/microglial activation in the pathogenesis.

BACKGROUND: The zitter (zi/zi) rat, a loss-of-function mutant of the glycosylated transmembrane protein attractin (atrn), exhibits widespread age-dependent spongiform degeneration, hypomyelination, and abnormal metabolism of reactive oxygen species (ROS) in the brain. To date, the mechanisms underlying these phenotypes have remained unclear. RESULTS: Here, we show differentiation defects in zi/zi oligodendrocytes, accompanied by aberrant extension of cell-processes and hypomyelination. Axonal bundles were relatively preserved during postnatal development. With increasing in age, the injured oligodendrocytes in zi/zi rats become pathological, as evidenced by the accumulation of iron in their cell bodies. Immunohistochemical analysis revealed that atrn expression was absent from an oligodendrocyte lineage, including A2B5-positive progenitors and CNPase-positive differentiated cells. The number and distribution of Olig2-positive oligodendrocyte progenitors was unchanged in the zi/zi brain. Furthermore, an in vitro differentiation assay of cultured oligodendrocyte progenitors prepared from zi/zi brains revealed their normal competence for proliferation and differentiation into mature oligodendrocytes. Interestingly, we demonstrated the accelerated recruitment of ED1-positive macrophages/microglia to the developing zi/zi brain parenchyma prior to the onset of hypomyelination. Semiquantitative RT-PCR analysis revealed a significant up-regulation of CD26 and IL1-beta in the zi/zi brain during this early postnatal stage. CONCLUSION: We demonstrated that the onset of the impairment of oligodendrocyte differentiation occurs in a non-cell autonomous manner in zi/zi rats. Hypomyelination of oligodendrocytes was not due to a failure of the intrinsic program of oligodendrocytes, but rather, was caused by extrinsic factors that interrupt oligodendrocyte development. It is likely that macrophage/microglial activation in the zi/zi CNS leads to disturbances in oligodendrocyte differentiation via deleterious extrinsic factors, such as the cytokine IL1-beta or ROS. Atrn might be involved in the activation of brain macrophages/microglia by suppressing excessive migration of monocytes into the CNS, or by accelerating the transformation of brain monocytes into resting microglia. Understanding the pathogenesis of the zi/zi rat may provide novel insights into the developmental interaction betweens macrophages/microglia and cells of an oligodendrocyte lineage.

Role of stress fibers and focal adhesions as a mediator for mechano-signal transduction in endothelial cells in situ.

Fluid shear stress is the mechanical force generated by the blood flow which is applied over the apical surface of endothelial cells in situ. The findings of a recent study suggest that stress fibers and its associated focal adhesions play roles in mechano-signal transduction mechanism. Stress fibers are present along the apical and the basal portion of the endothelial cells. Endothelial cells respond to fluid shear stress and change their morphological characteristics in both their cell shape and cytoskeletal organization. Atherosclerosis is a common disease of the arteries and it occurs in areas around the branching site of blood vessels where the cells are exposed to low fluid shear stress. The organization of stress fibers and focal adhesions are strongly influenced by shear stress, and therefore the generation of atherosclerotic lesions seem to be associated with the cytoskeletal components of endothelial cells. This review describes the possible role of the cytoskeleton as a mechano-transducer in endothelial cells in situ.

Six1 is essential for early neurogenesis in the development of olfactory epithelium.

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, "early neurogenesis" occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows "established neurogenesis," in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1-/-). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1-/- at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1-/-. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.

Rho-kinase dependent organization of stress fibers and focal adhesions in cultured fibroblasts.

The activation of Rho-kinase is known to modulate the organization of the actin-based cytoskeletal systems, including the formation of stress fibers and focal adhesions. Rho-kinase likely plays a more crucial and complex role in the organization of actin-based cytoskeletal systems than in that of myosin light chain kinase (MLCK). In order to understand the role of Rho-kinase in the organization of stress fibers and focal adhesions, we treated cultured fibroblasts with a Rho-kinase inhibitor and analyzed the stress fiber and focal adhesion organization under conventional fluorescence microscopy and replica electron microscopy. Some of the cells were transfected with GFP-labeled paxillin, actin or alpha-actinin, and the effects of the inhibitor were monitored in the living cells. The Rho-kinase inhibitor caused disassembly of the stress fibers and focal adhesions in the central portion of the cell within 1 h. However, the stress fibers and focal adhesions located in the cell periphery were not as severely affected by the Rho-kinase inhibitor. The time-lapse video recording revealed that when these cells were washed with a fresh medium in order to remove the Rho-kinase inhibitor, the stress fibers and focal adhesions located in the center of the cell gradually reorganized and, within 1.5-2 h, the cells completely recovered. This observation strongly suggests that the activation of Rho-kinase plays an important role in the organization of the central stress fibers and focal adhesions.

Morphological differences between guinea pig aortic and venous endothelial cells in situ.

Endothelial cells (ECs) respond to fluid shear stress. They reveal shear stress related morphological changes in both their cell shape and cytoskeletal organization. Little is known about the cytoskeletal organization of ECs in situ. We studied, together with the living ultrasound high resolution imaging system, the distribution of stress fibers (SFs), certain focal adhesion (FA) and signal transduction associated proteins in guinea pig aortic and venous ECs. Although SFs present in the basal portion of venous ECs ran along the direction of the blood flow, their size was smaller and their number was fewer than those of aortic ECs. Venous ECs were elongated to the direction of flow than in aortic ECs exposed over normal shear stress (SS). Since fluid SS in the vein is low, a sustained and uni-directional low SS over a long period might thus cause these structural features observed in venous ECs.

Testosterone-dependent transgene expression in the liver of the CAG-lacZ transgenic rat.

Many endogenous gene expressions in the liver are well known to be predominant in males, compared with those of females. In contrast, the fate of hepatic transgene expression between sexes is not fully understood. Here we studied whether sex hormones changed hepatic transgene expression in the ubiquitous CAG promoter-driven lacZ transgenic (Tg) rat. Both sexes of CAG-lacZ Tg rats received gonadectomy. Liver biopsy was taken weekly to determine the change of transgene expression. Histological result of adult males showed mosaic lacZ expression but it was negative in adult females, while livers in neonatal stage showed comparable expression of lacZ. Other organs exhibited equal expression in both sexes. At 2 weeks after castration, lacZ expression in male liver was significantly decreased and became negative after 4 weeks while no significant difference was observed in the lacZ expression pattern in other organs. After ovariectomy, lacZ expression in female liver remained undetectable. Moreover, testosterone treatment to gonadectomized rats of both sexes could enhance lacZ expression in the liver. In summary, we report that CAG-lacZ Tg rats demonstrate sexual dimorphism of transgene expression specifically only in the liver. Testosterone administration mediated upregulation of liver lacZ expression. Our findings suggested that androgen, especially testosterone, plays an important role in the hepatic transgene expression.

Disruption of aquaporin-11 produces polycystic kidneys following vacuolization of the proximal tubule.

Aquaporin-11 (AQP11) has been identified with unusual pore-forming NPA (asparagine-proline-alanine) boxes, but its function is unknown. We investigated its potential contribution to the kidney. Immunohistochemistry revealed that AQP11 was localized intracellularly in the proximal tubule. When AQP11 was transfected in CHO-K1 cells, it was localized in intracellular organelles. AQP11-null mice were generated; these mice exhibited vacuolization and cyst formation of the proximal tubule. AQP11-null mice were born normally but died before weaning due to advanced renal failure with polycystic kidneys, in which cysts occupied the whole cortex. Remarkably, cyst epithelia contained vacuoles. These vacuoles were present in the proximal tubules of newborn mice. In 3-week-old mice, these tubules contained multiple cysts. Primary cultured cells of the proximal tubule revealed an endosomal acidification defect in AQP11-null mice. These data demonstrate that AQP11 is essential for the proximal tubular function. AQP11-null mice are a novel model for polycystic kidney diseases and will provide a new mechanism for cystogenesis.

The effects of Levovist and DD-723 in activating platelets and damaging hepatic cells of rats.

OBJECTIVE: The purpose of this study was to compare platelet activation and hepatic cell damage produced by 2 ultrasonographic contrast agents with flow cytometric and ultrastructural analysis. METHODS: Suspension samples were made by mixing Levovist (SH U508A; Schering AG, Berlin, Germany) or DD-723 (Nycomed; Amersham Health, Princeton, NJ) with whole blood. The final concentrations of Levovist in citrated whole blood were 0, 15, and 75 mg/mL, and those of DD-723 were 0, 5, and 50 microL/mL. After exposure to ultrasound in vitro, flow cytometric analysis was performed to determine the concentration of the CD62P activation-specific antigen. To compare the hepatic cell damage associated with these 2 agents, we divided 15 rats into 5 groups as follows: group 1, sham operation; group 2, Levovist injection only; group 3, DD-723 injection only; group 4, Levovist injection (contrast agent) and ultrasound exposure; and group 5, DD-723 injection and ultrasound exposure. The ultrasonographic contrast agents Levovist and DD-723 were administered through the femoral vein and sonicated continuously for the first minute; this was followed by sweeping for 5 minutes 10 seconds after the contrast agent was injected. The rats were perfused via the heart with a fixative solution immediately after the sweeping, and then the liver was excised; the specimens were studied with electron and light microscopy. RESULTS: The percentage of CD62P-expressing platelets increased in both contrast agent-ultrasound exposure groups, and the percentage of CD62P-expressing platelets was greater in the Levovist group. We observed vacuolation and round deposits in the hepatocytes in both contrast agent-ultrasound exposure groups. Microbubbles were observed in the rat Kupffer cells, and a few hepatocytes were seen unexpectedly in the DD-723 group but were found in neither the Kupffer cells nor the hepatocytes in the Levovist group. CONCLUSIONS: Both contrast agents, Levovist and DD-723, produced platelet activation and structural change in the rat hepatic cells, but only the microbubbles of DD-723 were taken up by the Kupffer cells and a few hepatocytes.

Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation.

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.

Prevention of glomerular crescent formation in glomerulonephritis by mycophenolate mofetil in rats.

BACKGROUND: Glomerular crescent formation is a prominent feature of aggressive forms of glomerulonephritis (GN) and is associated with a poor prognosis. We investigated whether the potent immunosuppressive agent mycophenolate mofetil (MMF) could prevent crescent formation in a model of anti-glomerular basement membrane (GBM) GN in the rat. METHODS: GN with glomerular crescents was induced by the injection of anti-GBM antibody to female Wistar-Kyoto (WKY/NCrj) rats. The experimental rats were divided into two groups: rats received vehicle (0.5% carboxymethylcerlose) or MMF (20 mg/kg/day) orally. Body weight was measured and the urine and blood samples were evaluated. The rats were sacrificed at day 14, and histological analysis was performed. The mRNA expression of cytokines and adhesion molecules in the kidney was analysed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Marked proteinuria, glomerular crescent formation and glomerulosclerosis were observed in this model, and these were significantly reduced by MMF treatment. Marked glomerular macrophage and T-cell infiltration was also observed, and MMF treatment significantly inhibited macrophage but not T-cell infiltration. RT-PCR and immunohistochemical analysis revealed that mRNA and protein expression of osteopontin was decreased by the treatment with MMF. In addition, MMF treatment in the early stages of GN could inhibit proteinuria, glomerular crescent formation and glomerulosclerosis. CONCLUSIONS: These findings suggest therapeutic potential for MMF in the inhibition of glomerular crescent formation in GN and provide new insights into the mechanism underlying the amelioration of crescentic GN by MMF treatment.

Endothelial cell injury and platelet aggregation induced by contrast ultrasonography in the rat hepatic sinusoid.

OBJECTIVE: To determine whether contrast ultrasonography can affect the sinusoidal cells and platelets of the liver by using ultrastructural analysis in vivo. METHODS: Fifteen Wistar rats were placed into the following 5 groups of 3 rats each: 3 control groups comprising a sham operation group, a contrast agent injection-alone group, and an ultrasound exposure-alone group; and 2 contrast agent injection with ultrasound exposure groups, split according to excision time. After a dose of an echo contrast agent (100 mg/kg of body weight) was administered through the femoral vein, the rats that received injections were subjected to ultrasound for the first minute, no ultrasound for the next 4 minutes, and then ultrasound sweep scanning for 10 seconds. The rats were perfused via the heart with cold physiologic saline containing 2% paraformaldehyde and 2.5% glutaraldehyde solution buffered with 0.1-mol/L phosphate. The livers of the rats in 4 of the groups were excised immediately. The livers of the rats in 1 of the 2 contrast agent with ultrasound exposure groups were excised by the same procedure 5 hours after they received the injections. All specimens were studied with light and electron microscopy. RESULTS: Platelet aggregation and injury to endothelial cells were more severe in the contrast agent injection and ultrasound exposure groups than in the other groups. CONCLUSIONS: Contrast ultrasonography can cause platelet aggregation and endothelial cell damage in the rat hepatic sinusoid.

Localization of mechanosensitive channel TRPV4 in mouse skin.

A transient receptor potential (TRP) family, TRPV4, is a calcium-permeable swell-activated channel, playing a role in cutaneous mechanosensation. To elucidate the localization in the mechanosensitive endings, we found with immunohistochemistry in mice that TRPV4 was expressed both by small (low threshold) and large (high threshold) dorsal root ganglia neurons. In addition to free nerve endings, TRPV4 was specifically located at cutaneous mechanosensory terminals co-localized with neurofilament 200, including Meissner, Merkel, penicillate and intraepidermal terminals but not including hair follicle palisades. The distribution suggests that the sensation of pressure by mechanosensitive TRPV4 channel is transmitted through A- as well as C-fiber.

Engraftment and tumor formation after allogeneic in utero transplantation of primate embryonic stem cells.

BACKGROUND: To achieve human embryonic stem (ES) cell-based transplantation therapies, allogeneic transplantation models of nonhuman primates would be useful. We have prepared cynomolgus ES cells genetically marked with the green fluorescent protein (GFP). The cells were transplanted into the allogeneic fetus, taking advantage of the fact that the fetus is so immunologically immature as not to induce immune responses to transplanted cells and that fetal tissue compartments are rapidly expanding and thus providing space for the engraftment. METHODS: Cynomolgus ES cells were genetically modified to express the GFP gene using a simian immunodeficiency viral vector or electroporation. These cells were transplanted in utero with ultrasound guidance into the cynomolgus fetus in the abdominal cavity (n=2) or liver (n=2) at the end of the first trimester. Three fetuses were delivered 1 month after transplantation, and the other, 3 months after transplantation. Fetal tissues were examined for transplanted cell progeny by quantitative polymerase chain reaction and in situ polymerase chain reaction of the GFP sequence. RESULTS: A fluorescent tumor, obviously derived from transplanted ES cells, was found in the thoracic cavity at 3 months after transplantation in one fetus. However, transplanted cell progeny were also detected (approximately 1%) without teratomas in multiple fetal tissues. The cells were solitary and indistinguishable from surrounding host cells. CONCLUSIONS: Transplanted cynomolgus ES cells can be engrafted in allogeneic fetuses. The cells will, however, form a tumor if they "leak" into an improper space such as the thoracic cavity.

Morphologic characterization of green fluorescent protein in embryonic, neonatal, and adult transgenic rats.

Transgenic (Tg) animals with reporter genes are useful models in which to study cell lineage and the process of differentiation into tissues. We developed the green fluorescent protein (GFP)-Tg rat, which is more suitable for transplantation and stem cell research because it is larger than mice. We found that marker gene expression was dependent on each organ and developmental stage. In this study we describe GFP expression in various tissues from embryonic, neonatal, and adult animals. GFP expression in brain, lung, liver, and islet tissues was restricted to early developmental stages, but it was continuously strong in the exocrine pancreas, kidney, and cardiac and skeletal muscles. The CAG promoter that was presumed to induce ubiquitous protein expression might be responsible for the differences in expression.

Antiapoptotic effect of endothelin-1 in rat cardiomyocytes in vitro.

Apoptosis of cardiac myocytes is thought to be a feature of many pathological disorders, including congestive heart failure (CHF) and ischemic heart disease (IHD). Because recent investigations indicate that endothelin-1 (ET-1) plays an important role in CHF and IHD, we investigated the effect of ET-1 on cardiomyocyte apoptosis. The presence of apoptosis in rat cardiomyocytes (H9c2 and neonatal) was evaluated by morphological criteria, electrophoresis of DNA fragments, 4',6'-diamidine-2'-phenylindole staining, and TUNEL analysis. ET-1, but not angiotensin II, prevented apoptosis induced by serum deprivation via ETA receptors in a dose-dependent manner (1 to 100 nmol/L). ET-1 also prevented cytochrome c release from mitochondria to the cytosol. The use of specific pharmacological inhibitors demonstrated that the antiapoptotic effect of ET-1 was mediated through a tyrosine kinase pathway (genistein and AG490) but not through protein kinase C (PKC; calphostin C), mitogen-activated protein kinases (PD98059 and SB203580), or PKA (KT5270) pathways. Adenovirus-mediated gene transfer of kinase-inactive (KI) c-Src reversed the antiapoptotic effect of ET-1. We further investigated whether Bcl-xL, an antiapoptotic molecule, would be upregulated by using a luciferase-based reporter system. ET-1 upregulated Bcl-xL, and this upregulation was inhibited by genistein or AG490 but not by calphostin C. The experiments with KI mutants for various tyrosine kinases revealed that c-Src and Pyk2 (but not JAK1, Jak2, Syk, and Tec) are involved in ET-1-induced upregulation of Bcl-xL expression. These findings suggest that ET-1 prevents apoptosis in cardiac myocytes through the ETA receptor and the subsequent c-Src/Bcl-xL-dependent pathway.

Fluvastatin induces apoptosis in rat neonatal cardiac myocytes: a possible mechanism of statin-attenuated cardiac hypertrophy.

Hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) have been shown to reduce atherosclerotic cardiovascular mortality and morbidity. Recent evidence indicates that statins may also exert direct effects on vascular wall cells (including endothelial cells and smooth muscle cells) independently of their hypocholesterolemic properties. However, little is known about whether statins have direct effects on myocardium. The effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis and protein synthesis in rat neonatal cardiac myocytes was investigated. The presence of apoptosis was evaluated by morphologic criteria, electrophoresis of DNA fragments, 4",6"-diamidine-2"-phenylindole (DAPI) staining, and TUNEL assay. Protein synthesis was measured by H-leucine incorporation into the cells. Fluvastatin, but not pravastatin, induced apoptosis in cardiac myocytes in a time- and dose-dependent manner. The pro-apoptotic effect of fluvastatin was reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate (GGPP), but not in the presence of squalene. The addition of protein prenylation inhibitor perillic acid and Rho-kinase inhibitor Y27632 significantly increased apoptosis. Fluvastatin decreased RhoA protein in the membrane fraction, whereas there were no significant changes of the RhoA protein in the cytosol fraction. Interleukin-1beta-stimulated H-leucine incorporation was completely inhibited by fluvastatin, but not by pravastatin. The findings suggest that fluvastatin induces apoptosis in cardiac myocytes via protein prenylation and the subsequent inhibition of Rho, and may play a role in the pathogenesis of cardiac hypertrophy and remodeling.