Essential role of Cdc42 in cardiomyocyte proliferation and cell-cell adhesion during heart development.

Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/ß-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development.

miR-24 Inhibition Increases Menin Expression and Decreases Cholangiocarcinoma Proliferation.

Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24-dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.

Nicotine Promotes Cholangiocarcinoma Growth in Xenograft Mice.

Nicotine, the main addictive substance in tobacco, is known to play a role in the development and/or progression of a number of malignant tumors. However, nicotine's involvement in the pathogenesis of cholangiocarcinoma is controversial. Therefore, we studied the effects of nicotine on the growth of cholangiocarcinoma cells in vitro and the progression of cholangiocarcinoma in a mouse xenograft model. The predominant subunit responsible for nicotine-mediated proliferation in normal and cancer cells, the α7 nicotinic acetylcholine receptor (α7-nAChR), was more highly expressed in human cholangiocarcinoma cell lines compared with normal human cholangiocytes. Nicotine also stimulated the proliferation of cholangiocarcinoma cell lines and promoted α7-nAChR-dependent activation of proliferation and phosphorylation of extracellular-regulated kinase in Mz-ChA-1 cells. In addition, nicotine and PNU282987 (α7-nAChR agonist) accelerated the growth of the cholangiocarcinoma tumors in our xenograft mouse model and increased fibrosis, proliferation of the tumor cells, and phosphorylation of extracellular-regulated kinase activation. Finally, α7-nAChR was expressed at significantly higher levels in human cholangiocarcinoma compared with normal human control liver samples. Taken together, results of this study suggest that nicotine acts through α7-nAChR and plays a novel role in the pathogenesis of cholangiocarcinoma. Furthermore, nicotine may act as a mitogen in cholestatic liver disease processes, thereby facilitating malignant transformation.

α7-nAChR Knockout Mice Decreases Biliary Hyperplasia and Liver Fibrosis in Cholestatic Bile Duct-Ligated Mice.

α7-nAChR is a nicotinic acetylcholine receptor [specifically expressed on hepatic stellate cells (HSCs), Kupffer cells, and cholangiocytes] that regulates inflammation and apoptosis in the liver. Thus, targeting α7-nAChR may be therapeutic in biliary diseases. Bile duct ligation (BDL) was performed on wild-type (WT) and α7-nAChR-/- mice. We first evaluated the expression of α7-nAChR by immunohistochemistry (IHC) in liver sections. IHC was also performed to assess intrahepatic bile duct mass (IBDM), and Sirius Red staining was performed to quantify the amount of collagen deposition. Immunofluorescence was performed to assess colocalization of α7-nAChR with bile ducts (costained with CK-19) and HSCs (costained with desmin). The mRNA expression of α7-nAChR, Ki-67/PCNA (proliferation), fibrosis genes (TGF-ß1, fibronectin-1, Col1α1, and α-SMA), and inflammatory markers (IL-6, IL-1ß, and TNF-α) was measured by real-time PCR. Biliary TGF-ß1 and hepatic CD68 (Kupffer cell marker) expression was assessed using IHC. α7-nAChR immunoreactivity was observed in both bile ducts and HSCs and increased following BDL. α7-nAChR-/- BDL mice exhibited decreased (i) bile duct mass, liver fibrosis, and inflammation, and (ii) immunoreactivity of TGF-ß1 as well as expression of fibrosis genes compared to WT BDL mice. α7-nAChR activation triggers biliary proliferation and liver fibrosis and may be a therapeutic target in managing extrahepatic biliary obstruction.

Loss of myocardial retinoic acid receptor α induces diastolic dysfunction by promoting intracellular oxidative stress and calcium mishandling in adult mice.

Retinoic acid receptor (RAR) has been implicated in pathological stimuli-induced cardiac remodeling. To determine whether the impairment of RARα signaling directly contributes to the development of heart dysfunction and the involved mechanisms, tamoxifen-induced myocardial specific RARα deletion (RARαKO) mice were utilized. Echocardiographic and cardiac catheterization studies showed significant diastolic dysfunction after 16wks of gene deletion. However, no significant differences were observed in left ventricular ejection fraction (LVEF), between RARαKO and wild type (WT) control mice. DHE staining showed increased intracellular reactive oxygen species (ROS) generation in the hearts of RARαKO mice. Significantly increased NOX2 (NADPH oxidase 2) and NOX4 levels and decreased SOD1 and SOD2 levels were observed in RARαKO mouse hearts, which were rescued by overexpression of RARα in cardiomyocytes. Decreased SERCA2a expression and phosphorylation of phospholamban (PLB), along with decreased phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) was observed in RARαKO mouse hearts. Ca2+ reuptake and cardiomyocyte relaxation were delayed by RARα deletion. Overexpression of RARα or inhibition of ROS generation or NOX activation prevented RARα deletion-induced decrease in SERCA2a expression/activation and delayed Ca2+ reuptake. Moreover, the gene and protein expression of RARα was significantly decreased in aged or metabolic stressed mouse hearts. RARα deletion accelerated the development of diastolic dysfunction in streptozotocin (STZ)-induced type 1 diabetic mice or in high fat diet fed mice. In conclusion, myocardial RARα deletion promoted diastolic dysfunction, with a relative preserved LVEF. Increased oxidative stress have an important role in the decreased expression/activation of SERCA2a and Ca2+ mishandling in RARαKO mice, which are major contributing factors in the development of diastolic dysfunction. These data suggest that impairment of cardiac RARα signaling may be a novel mechanism that is directly linked to pathological stimuli-induced diastolic dysfunction.

Activation of the renin-angiotensin system stimulates biliary hyperplasia during cholestasis induced by extrahepatic bile duct ligation.

Cholangiocyte proliferation is regulated in a coordinated fashion by many neuroendocrine factors through autocrine and paracrine mechanisms. The renin-angiotensin system (RAS) is known to play a role in the activation of hepatic stellate cells and blocking the RAS attenuates hepatic fibrosis. We investigated the role of the RAS during extrahepatic cholestasis induced by bile duct ligation (BDL). In this study, we used normal and BDL rats that were treated with control, angiotensin II (ANG II), or losartan for 2 wk. In vitro studies were performed in a primary rat cholangiocyte cell line (NRIC). The expression of renin, angiotensin-converting enzyme, angiotensinogen, and angiotensin receptor type 1 was evaluated by immunohistochemistry (IHC), real-time PCR, and FACs and found to be increased in BDL compared with normal rat. The levels of ANG II were evaluated by ELISA and found to be increased in serum and conditioned media of cholangiocytes from BDL compared with normal rats. Treatment with ANG II increased biliary mass and proliferation in both normal and BDL rats. Losartan attenuated BDL-induced biliary proliferation. In vitro, ANG II stimulated NRIC proliferation via increased intracellular cAMP levels and activation of the PKA/ERK/CREB intracellular signaling pathway. ANG II stimulated a significant increase in Sirius red staining and IHC for fibronectin that was blocked by angiotensin receptor blockade. In vitro, ANG II stimulated the gene expression of collagen 1A1, fibronectin 1, and IL-6. These results indicate that cholangiocytes express a local RAS and that ANG II plays an important role in regulating biliary proliferation and fibrosis during extraheptic cholestasis.

General mechanisms of nicotine-induced fibrogenesis.

Cigarette smoking contributes to the development of cancer, and pathogenesis of other diseases. Many chemicals have been identified in cigarettes that have potent biological properties. Nicotine is especially known for its role in addiction and plays a role in other physiological effects of smoking and tobacco use. Recent studies have provided compelling evidence that, in addition to promoting cancer, nicotine also plays a pathogenic role in systems, such as the lung, kidney, heart, and liver. In many organ systems, nicotine modulates fibrosis by altering the functions of fibroblasts. Understanding the processes modulated by nicotine holds therapeutic potential and may guide future clinical and research decisions. This review discusses the role of nicotine in the general fibrogenic process that governs fibrosis and fibrosis-related diseases, focusing on the cellular mechanisms that have implications in multiple organ systems. Potential research directions for the management of nicotine-induced fibrosis, and potential clinical considerations with regard to nicotine-replacement therapy (NRT) are presented.

Cardiac dysfunction subsequent to chronic ozone exposure in rats.

A number of advancements have been made toward identifying the risk factors associated with cardiovascular disease (CVD) and have resulted in a decline in mortality. However, many patients with cardiac disease show no established previous risk. Thus, it appears that other unknown factors contribute to the pathophysiology of CVD. Out of 350,000 sudden cardiac deaths each year in the United States, 60,000 deaths have been linked to air pollution, suggesting a detrimental role of environmental pollutants in the development of CVD. This study tested the hypothesis that chronic ozone (O(3)) exposure diminishes myocardial function in healthy population. Male Sprague-Dawley rats were exposed 8 h/day for 28 and 56 days to filtered air or 0.8 ppm O(3). In vivo cardiac function was assessed by measuring LVDP, +dP/dt, -dP/dt, and LVEDP 24 h after termination of the O(3) exposure. Compared to rats exposed to filtered air, LVDP, +dP/dt, and -dP/dt were significantly decreased, and LVEDP was significantly increased in O(3) exposed animals. This attenuation of cardiac function was associated with increased myocardial TNF-alpha levels and lipid peroxidation as well as decreased myocardial activities of superoxidase dismutase and interleukin-10 levels. These novel findings suggest myocardial dysfunction subsequent to chronic O(3) exposure in normal adult rats may be associated with a decrease in antioxidant reserve and with an increased production of inflammatory mediators.

Differential expression of caveolin-1 and caveolin-3: potential marker for cardiac toxicity subsequent to chronic ozone inhalation.

Earlier studies from our laboratory have shown myocardial dysfunction subsequent to chronic O(3) exposure in rats may be associated with a decrease in antioxidant reserve and increased activity of inflammatory mediators. The present study tested the hypothesis that O(3)-induced cardiac dysfunction in healthy adult rats may be due to changes in caveolin-1 and caveolin-3 levels. Sprague-Dawley rats were exposed 8 h/day for 28 and 56 days to filtered air or 0.8 ppm O(3). In order to assess the chronic effects to O(3), in vivo cardiac function was assessed by measuring LVDP, 24 h after termination of O(3) exposure. Compared to rats exposed to filtered air, LVDP values significantly decreased in all O(3)-exposed animals. This attenuation of cardiac function was associated with increased myocardial TNF-α levels and decreased myocardial activities of superoxidase dismutase. Progressive increases in the expression of myocardial TNF-α in 28 days and 56 days O(3)-exposed animals were followed by decreases in cardiac caveolin-1 levels. On the other hand, differential changes in the expression of caveolin-3 in hearts from 28 and 56 days O(3)-exposed animals were independent of intra-cardiac TNF-α levels. These novel findings suggest the interesting possibility that a balance between caveolin-1 and caveolin-3 may be involved in O(3)-mediated cardiac toxicity.

Estrogen Protects Cardiac Function and Energy Metabolism in Dilated Cardiomyopathy Induced by Loss of Cardiac IRS1 and IRS2.

BACKGROUND: Type 2 diabetes (T2D) is a high-risk factor for incident of cardiovascular diseases. Women at young ages show a reduced incidence of both T2D and cardiovascular diseases compared with men, but these disparities disappear in postmenopausal women versus age-matched men. Thus, ovaries and ovarian hormones, such as estrogen, are expected to protect from T2D and cardiovascular diseases. In this study, we aimed to investigate the role of ovaries and ovarian hormone estrogen in cardiac function and energy metabolism using the cardiac IRS (insulin receptor substrate) 1 and IRS2 double genes knockout mice that mimic cardiac insulin resistance. METHODS: Control and heart-specific IRS1/2 double genes knockout mice were treated with placebo or 17ß-estradiol (E2) pellets, respectively, through subcutaneous implantation. Female mice were subjected to a bilateral ovariectomy surgery to remove endogenous E2. The cardiac function and energy metabolism were determined using echocardiography and indirect calorimeter, respectively. RESULTS: All male heart-specific IRS1/2 double genes knockout mice died of heart failure at 6 to 8 weeks as we previously described (Qi et al), but all female heart-specific IRS1/2 double genes knockout mice survived >1 year. Removal of ovaries in heart-specific IRS1/2 double genes knockout female mice resulted in cardiac dysfunction, and ultimately animal death. However, E2 supplementation prevented the dilated cardiomyopathy, improved cardiac function and energy metabolism, and enhanced lifespan in both male and ovariectomy female mice deficient for cardiac IRS1 and IRS2 genes, largely owing to the activation of Akt (protein kinase B)-Foxo1 (O1 class of forkhead/winged helix transcription factor) signaling cascades. CONCLUSIONS: These results show that estrogen protects mice from cardiac insulin resistance-induced diabetic cardiomyopathy. This may provide a fundamental mechanism for the gender difference for the incidence of both T2D and cardiovascular diseases. This study highlights that estrogen signaling could be a potential target for improving cardiac function and energy metabolism in humans with T2D.

Castration inhibits biliary proliferation induced by bile duct obstruction: novel role for the autocrine trophic effect of testosterone.

Increased cholangiocyte growth is critical for the maintenance of biliary mass during liver injury by bile duct ligation (BDL). Circulating levels of testosterone decline following castration and during cholestasis. Cholangiocytes secrete sex hormones sustaining cholangiocyte growth by autocrine mechanisms. We tested the hypothesis that testosterone is an autocrine trophic factor stimulating biliary growth. The expression of androgen receptor (AR) was determined in liver sections, male cholangiocytes, and cholangiocyte cultures [normal rat intrahepatic cholangiocyte cultures (NRICC)]. Normal or BDL (immediately after surgery) rats were treated with testosterone or antitestosterone antibody or underwent surgical castration (followed by administration of testosterone) for 1 wk. We evaluated testosterone serum levels; intrahepatic bile duct mass (IBDM) in liver sections of female and male rats following the administration of testosterone; and secretin-stimulated cAMP levels and bile secretion. We evaluated the expression of 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3, the enzyme regulating testosterone synthesis) in cholangiocytes. We evaluated the effect of testosterone on the proliferation of NRICC in the absence/presence of flutamide (AR antagonist) and antitestosterone antibody and the expression of 17ß-HSD3. Proliferation of NRICC was evaluated following stable knock down of 17ß-HSD3. We found that cholangiocytes and NRICC expressed AR. Testosterone serum levels decreased in castrated rats (prevented by the administration of testosterone) and rats receiving antitestosterone antibody. Castration decreased IBDM and secretin-stimulated cAMP levels and ductal secretion of BDL rats. Testosterone increased 17ß-HSD3 expression and proliferation in NRICC that was blocked by flutamide and antitestosterone antibody. Knock down of 17ß-HSD3 blocks the proliferation of NRICC. Drug targeting of 17ß-HSD3 may be important for managing cholangiopathies.

Knockout of secretin receptor reduces large cholangiocyte hyperplasia in mice with extrahepatic cholestasis induced by bile duct ligation.

UNLABELLED: During bile duct ligation (BDL), the growth of large cholangiocytes is regulated by the cyclic adenosine monophosphate (cAMP)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and is closely associated with increased secretin receptor (SR) expression. Although it has been suggested that SR modulates cholangiocyte growth, direct evidence for secretin-dependent proliferation is lacking. SR wild-type (WT) (SR(+/+)) or SR knockout (SR(-/-)) mice underwent sham surgery or BDL for 3 or 7 days. We evaluated SR expression, cholangiocyte proliferation, and apoptosis in liver sections and proliferating cell nuclear antigen (PCNA) protein expression and ERK1/2 phosphorylation in purified large cholangiocytes from WT and SR(-/-) BDL mice. Normal WT mice were treated with secretin (2.5 nmoles/kg/day by way of osmotic minipumps for 1 week), and biliary mass was evaluated. Small and large cholangiocytes were used to evaluate the in vitro effect of secretin (100 nM) on proliferation, protein kinase A (PKA) activity, and ERK1/2 phosphorylation. SR expression was also stably knocked down by short hairpin RNA, and basal and secretin-stimulated cAMP levels (a functional index of biliary growth) and proliferation were determined. SR was expressed by large cholangiocytes. Knockout of SR significantly decreased large cholangiocyte growth induced by BDL, which was associated with enhanced apoptosis. PCNA expression and ERK1/2 phosphorylation were decreased in large cholangiocytes from SR(-/-) BDL compared with WT BDL mice. In vivo administration of secretin to normal WT mice increased ductal mass. In vitro, secretin increased proliferation, PKA activity, and ERK1/2 phosphorylation of large cholangiocytes that was blocked by PKA and mitogen-activated protein kinase kinase inhibitors. Stable knockdown of SR expression reduced basal cholangiocyte proliferation. SR is an important trophic regulator sustaining biliary growth. CONCLUSION: The current study provides strong support for the potential use of secretin as a therapy for ductopenic liver diseases.

Isolation of Adult Mouse Cardiomyocytes Using Langendorff Perfusion Apparatus.

Heart disease is one of the leading causes of death in the United States. Isolation and culture adult cardiomyocytes are important for studying cardiomyocyte contractility, heart hypertrophy, and cardiac failure. In contrast to neonatal cardiomyocyte isolation, adult mice cardiomyocytes isolation is challenging due to firm connections among cardiomyocytes through intercalated discs. The availability of newly generated genetically modified mouse lines requires to establish protocols to isolation and culture adult mouse cardiomyocyte for in vitro studies. In this manuscript, we described a straightforward method of isolating adult mouse cardiomyocytes using Langendorff perfusion apparatus. Briefly, the hearts were harvested from adult mice and the heart was mounted to Lagendorff apparatus. After perfusion with calcium depletion and collagenase digestion, the left ventricles were minced and filtered. Lastly, the separated cardiomyocytes were treated with CaCl2. The isolated cardiac myocytes can be utilized in a broad range of experiments including screening for drugs.

Preparation of Neonatal Rat Papillary Muscles for Contractile Studies.

Isolated cardiac tissue allows investigators to study mechanisms underlying normal and pathological conditions, which would otherwise be difficult or impossible to perform in vivo. In contrast to ventricular muscle strip preparations, papillary muscles can be prepared without severely damaging the muscle tissue. In this preparation, the isolated papillary muscle is fixed in an environmentally controlled organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer, and parameters such as twitch force amplitude and twitch kinetics are analyzed. A variety of experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents, as well as simulation of pathologic conditions such as acute cardiac ischemia. Mouse papillary muscle preparations have long been the mainstay for studying interactions between intracellular calcium regulation and contractile responses under a number of simulated pathophysiological conditions. These studies are often used to complement in vitro studies performed using isolated neonatal rat cardiac myocytes. In this procedure, we describe how neonatal rat papillary muscles can also be prepared for use in contractile studies.

Secretin inhibits cholangiocarcinoma growth via dysregulation of the cAMP-dependent signaling mechanisms of secretin receptor.

Secretin plays a key role in the regulation of normal cholangiocyte physiology via secretin receptor (SCTR). SCTR expression is upregulated during extrahepatic cholestasis induced by bile duct ligation and closely associated with cholangiocyte proliferative responses. Although well studied in normal cholangiocytes, the role of secretin and the expression of SCTR in the regulation of cholangiocarcinoma proliferation are unknown. In vitro, secretin (10(-7) M) displayed differential effects on normal cholangiocyte [H-69 and human intrahepatic biliary epithelial cell line (HIBEpiC)] and cholangiocarcinoma (Mz-ChA-1, HuH-28, TFK-1, SG231, CCLP1 and HuCC-T1) cell lines as such secretin is mitogenic for normal cholangiocytes and antiproliferative for cholangiocarcinoma. As expected in normal cholangiocytes (HIBEpiC), secretin increased intracellular cyclic adenosine monophosphate (cAMP) levels. However, the effect of secretin on intracellular cAMP levels was suppressed in Mz-ChA-1 cells. Secretin-stimulated intracellular cAMP levels in Mz-ChA-1 were restored by pretreatment with pertussis toxin, suggesting that the receptor coupled to Galpha(i) rather than Galpha(s). SCTR expression was found to be downregulated in 4 of the 6 cholangiocarcinoma cell lines evaluated and in human cholangiocarcinoma biopsy samples. In vivo, secretin significantly inhibited the tumor size and more than doubled tumor latency, which was associated with a decrease in proliferating cell nuclear antigen and an increase in cleaved-caspase 3 expression levels. Our results demonstrate that secretin and/or the modulation of SCTR expression might have potential as a therapeutic tool in the treatment of cholangiocarcinoma.

Enhanced death signaling in ozone-exposed ischemic-reperfused hearts.

Although numerous advancements made in the field of human health have resulted in reduced deaths due to cardiovascular diseases (CVD), many patients with cardiac disease show no established risk. Therefore, other unknown factors may be responsible for the pathophysiology of CVD. Out of 350,000 sudden cardiac deaths each year in the United States, 60,000 deaths have been related to air pollution, suggesting a detrimental role of environmental pollutants in the development of CVD. The present study tested our hypothesis that chronic ozone exposure enhances the sensitivity to ischemia-reperfusion (I/R) injury in isolated perfused hearts. Sprague-Dawley rats were continuously exposed for 8 h/day for 28 and 56 days to filtered air or 0.8 ppm ozone. Isolated hearts were subjected to 30 min of global ischemia followed by 60 min of reperfusion. Cardiac function after I/R measured as left ventricular developed pressure (LVDP), +dP/dt, -dP/dt, and left ventricular end diastolic pressure (LVEDP) was significantly decreased and increased respectively in ozone-exposed I/R hearts compared to I/R hearts exposed to filtered air. The enhanced sensitivity to I/R injury upon ozone exposure was associated with increased myocardial TNF-alpha levels and lipid peroxidation and decreased myocardial activities of superoxidase dismutase (SOD) and IL-10. These data suggest that ozone-induced sensitivity to myocardial I/R injury may be due to promoting levels of oxidative stress as well as inflammatory mediators.

Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium.

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2-4 microm(2), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-) AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl(-)/HCO(3)(-) exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

Stretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: opposing roles of JNK1/2 and p38alpha MAP kinases.

The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy, remodeling, and fibroblast proliferation in the hemodynamically overloaded heart. However, the intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a substrate of the RAS system, are largely unknown. Here we report the identification of JNK1/2 as a negative, and p38alpha as a major positive regulator of Ao gene expression. Isolated neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB) plated on deformable membranes coated with collagen IV, were exposed to 20% equiaxial static-stretch (0-24 h). Mechanical stretch initially depressed Ao gene expression (4 h), whereas after 8 h, Ao gene expression increased in a time-dependent manner. Blockade of JNK1/2 with SP600125 increased basal Ao gene expression in NRVM (10.52+/-1.98 fold, P<0.001) and NRFB (13.32+/-2.07 fold, P<0.001). Adenovirus-mediated expression of wild-type JNK1 significantly inhibited, whereas expression of dominant-negative JNK1 and JNK2 increased basal and stretch-mediated (24 h) Ao gene expression, showing both JNK1 and JNK2 to be negative regulators of Ao gene expression in NRVM and NRFB. Blockade of p38alpha/beta by SB202190 or p38alpha by SB203580 significantly inhibited stretch-induced (24 h) Ao gene expression, whereas expression of wild-type p38alpha increased stretch-induced Ao gene expression in both NRVM (8.41+/-1.50 fold, P<0.001) and NRFB (3.39+/-0.74 fold, P<0.001). Conversely, expression of dominant-negative p38alpha significantly inhibited stretch response. Moreover, expression of constitutively active MKK6b (E) significantly stimulated Ao gene expression in the absence of stretch, indicating that p38 activation alone is sufficient to induce Ao gene expression. Taken together p38alpha was demonstrated to be a positive regulator, whereas JNK1/2 was found to be a negative regulator of Ao gene expression. Prolonged stretch diminished JNK1/2 activation, which was accompanied by a reciprocal increase in p38 activation and Ao gene expression. This suggests that a balance in JNK1/2 and p38alpha activation determines the level of Ao gene expression in myocardial cells.

Lethal and edema toxins of anthrax induce distinct hemodynamic dysfunction.

Fatalities due to anthrax are associated with severe hypotension suggesting that the toxins generated from Bacillus anthracis, lethal toxin (LeTx) and edema toxin (EdTx), have cardiovascular effects. Here, we demonstrate the effects of these toxins and characterize their effects by echocardiography. LeTx leads to a significant reduction in ejection fraction, decreased velocity of propagation (diastolic dysfunction), decreased velocity of circumferential fiber shortening (decreased contractility), and increased LV systolic area (pathophysiology). EdTx leads to a significant reduction in left ventricular volumes and cardiac output (reduced stroke volume) but does not cause significant change in ejection fraction or contractility. These results indicate that LeTx reduces left ventricular systolic function and EdTx reduces preload but does not have direct myocardial effects. Together, these findings suggest that LeTx and EdTx exert distinct hemodynamic dysfunction associated with anthrax infection.

Isolated neonatal rat papillary muscles: a new model to translate neonatal rat myocyte signaling into contractile mechanics.

Isolated cardiac tissue allows investigators to study mechanisms underlying normal and pathological conditions, which would otherwise be difficult or impossible to perform in vivo. Cultured neonatal rat ventricular cardiac myocytes (NRVM) are widely used to study signaling and growth mechanisms in the heart, primarily due to the versatility, economy, and convenience of this in vitro model. However, the lack of a well-defined longitudinal cellular axis greatly hampers the ability to measure contractile function in these cells, and therefore to associate signaling with mechanical function. In these methods, we demonstrate that this limitation can be overcome by using papillary muscles isolated from neonatal rat hearts. In the methods we describe procedures for isolation of right ventricular papillary muscles from 3-day-old neonatal rats and effects of mechanical and humoral stimuli on contraction and relaxation properties of these tissues.