Damir Nizamutdinov

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.—Jensen, K., Nizamutdinov, D., Guerrier, M., Afroze, S., Dostal, D., Glaser, S. General mechanisms of nicotine‐induced fibrogenesis. FASEB J. 26, 4778–4787 (2012). www.fasebj.org

Chronic nicotine exposure stimulates biliary growth and fibrosis in normal rats

BACKGROUND Epidemiological studies have indicated smoking to be a risk factor for the progression of liver diseases. Nicotine is the chief addictive substance in cigarette smoke and has powerful biological properties throughout the body. Nicotine has been implicated in a number of disease processes, including increased cell proliferation and fibrosis in several organ systems. AIMS The aim of this study was to evaluate the effects of chronic administration of nicotine on biliary proliferation and fibrosis in normal rats. METHODS In vivo, rats were treated with nicotine by osmotic minipumps for two weeks. Proliferation, α7-nicotinic receptor and profibrotic expression were evaluated in liver tissue, cholangiocytes and a polarized cholangiocyte cell line (normal rat intrahepatic cholangiocyte). Nicotine-dependent activation of the Ca(2+)/IP3/ERK 1/2 intracellular signalling pathway was also evaluated in normal rat intrahepatic cholangiocyte. RESULTS Cholangiocytes express α7-nicotinic receptor. Chronic administration of nicotine to normal rats stimulated biliary proliferation and profibrotic gene and protein expression such as alpha-smooth muscle actin and fibronectin 1. Activation of α7-nicotinic receptor stimulated Ca(2+)/ERK1/2-dependent cholangiocyte proliferation. CONCLUSION Chronic exposure to nicotine contributes to biliary fibrosis by activation of cholangiocyte proliferation and expression of profibrotic genes. Modulation of α7-nicotinic receptor signalling axis may be useful for the management of biliary proliferation and fibrosis during cholangiopathies.

Overview of Traumatic Brain Injury: An Immunological Context

Traumatic brain injury (TBI) afflicts people of all ages and genders, and the severity of injury ranges from concussion/mild TBI to severe TBI. Across all spectrums, TBI has wide-ranging, and variable symptomology and outcomes. Treatment options are lacking for the early neuropathology associated with TBIs and for the chronic neuropathological and neurobehavioral deficits. Inflammation and neuroinflammation appear to be major mediators of TBI outcomes. These systems are being intensively studies using animal models and human translational studies, in the hopes of understanding the mechanisms of TBI, and developing therapeutic strategies to improve the outcomes of the millions of people impacted by TBIs each year. This manuscript provides an overview of the epidemiology and outcomes of TBI, and presents data obtained from animal and human studies focusing on an inflammatory and immunological context. Such a context is timely, as recent studies blur the traditional understanding of an “immune-privileged” central nervous system. In presenting the evidence for specific, adaptive immune response after TBI, it is hoped that future studies will be interpreted using a broader perspective that includes the contributions of the peripheral immune system, to central nervous system disorders, notably TBI and post-traumatic syndromes.

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.

Production of Spontaneously Beating Neonatal Rat Heart Tissue for Calcium and Contractile Studies

Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FB) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Typically, these cell types are separated using Percoll density gradient procedures. Cells located between the Percoll bands (interband cells [IBCs]), which contain less mature NRVM and a variety of non-myocytes, including coronary vascular smooth muscle cells and endothelial cells (ECs), are routinely discarded. However, we have demonstrated that IBCs readily attach to extracellular matrix-coated coverslips, plastic culture dishes, and deformable membranes to form a 2-dimensional cardiac tissue layer which quickly develops spontaneous contraction within 24 h, providing a robust coculture model for the study of cell-to-cell signaling and contractile studies. Below, we describe methods that provide good cell yield and viability of IBCs during isolation of NRVM and FB obtained from 0- to 3-day-old neonatal rat pups. Basic characterization of IBCs and methods for use in intracellular calcium and contractile experiments are also presented. This method maximizes the use of cells obtained from neonatal rat hearts.

Caveolin and β1-integrin coordinate angiotensinogen expression in cardiac myocytes.

BACKGROUND The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy and remodeling, although the biochemical mechanisms responsible for regulating the local RAS are poorly understood. Caveolin-1 (Cav-1)/Cav-3 double-knockout mice display cardiac hypertrophy, and in vitro disruption of lipid rafts/caveolae using methyl-β-cyclodextrin (MβCD) abolishes cardiac protection. METHODS In this study, neonatal rat ventricular myocytes (NRVM) were used to determine whether lipid rafts/caveolae may be involved in the regulation of angiotensinogen (Ao) gene expression, a substrate of the RAS system. RESULTS Treatment with MβCD caused a time-dependent upregulation of Ao gene expression, which was associated with differential regulation of mitogen-activated protein (MAP) kinases ERK1/2, p38 and JNK phosphorylation. JNK was highly phosphorylated shortly after MβCD treatment (2-30 min), whereas marked activation of ERK1/2 and p38 occurred much later (2-4h). β1D-Integrin was required for MβCD-induced activation of the MAP kinases. Pharmacologic inhibition of ERK1/2 and JNK enhanced MβCD-induced Ao gene expression, whereas p38 blockade inhibited this response. Adenovirus-mediated expression of wild-type p38α enhanced MβCD-induced Ao gene expression; conversely expression of dominant negative p38α blocked the stimulatory effects of MβCD. Expression of Cav-3 siRNA stimulated Ao gene expression, whereas overexpression of Cav-3 was inhibitory. Cav-1 and Cav-3 expression levels were found to be positively regulated by p38, but unaffected by ERK1/2 and JNK. CONCLUSION Collectively, these studies indicate that lipid rafts/caveolae couple to Ao gene expression through a mechanism that involves β1-integrin and the differential actions of MAP kinase family members.

Anthrax lethal toxin induces acute diastolic dysfunction in rats through disruption of the phospholamban signaling network

BACKGROUND Anthrax lethal toxin (LT), secreted by Bacillus anthracis, causes severe cardiac dysfunction by unknown mechanisms. LT specifically cleaves the docking domains of MAPKK (MEKs); thus, we hypothesized that LT directly impairs cardiac function through dysregulation of MAPK signaling mechanisms. METHODS AND RESULTS In a time-course study of LT toxicity, echocardiography revealed acute diastolic heart failure accompanied by pulmonary regurgitation and left atrial dilation in adult Sprague-Dawley rats at time points corresponding to dysregulated JNK, phospholamban (PLB) and protein phosphatase 2A (PP2A) myocardial signaling. Using isolated rat ventricular myocytes, we identified the MEK7-JNK1-PP2A-PLB signaling axis to be important for regulation of intracellular calcium (Ca(2+)(i)) handling, PP2A activation and targeting of PP2A-B56α to Ca(2+)(i) handling proteins, such as PLB. Through a combination of gain-of-function and loss-of-function studies, we demonstrated that over-expression of MEK7 protects against LT-induced PP2A activation and Ca(2+)(i) dysregulation through activation of JNK1. Moreover, targeted phosphorylation of PLB-Thr(17) by Akt improved sarcoplasmic reticulum Ca(2+)(i) release and reuptake during LT toxicity. Co-immunoprecipitation experiments further revealed the pivotal role of MEK7-JNK-Akt complex formation for phosphorylation of PLB-Thr(17) during acute LT toxicity. CONCLUSIONS Our findings support a cardiogenic mechanism of LT-induced diastolic dysfunction, by which LT disrupts JNK1 signaling and results in Ca(2+)(i) dysregulation through diminished phosphorylation of PLB by Akt and increased dephosphorylation of PLB by PP2A. Integration of the MEK7-JNK1 signaling module with Akt represents an important stress-activated signalosome that may confer protection to sustain cardiac contractility and maintain normal levels of Ca(2+)(i) through PLB-T(17) phosphorylation.

Mechanosensing and regulation of cardiac function

The role of mechanical force as an important regulator of structure and function of mammalian cells, tissues, and organs has recently been recognized. However, mechanical overload is a pathogenesis or comorbidity existing in a variety of heart diseases, such as hypertension, aortic regurgitation and myocardial infarction. Physical stimuli sensed by cells are transmitted through intracellular signal transduction pathways resulting in altered physiological responses or pathological conditions. Emerging evidence from experimental studies indicate that β1-integrin and the angiotensin II type I (AT1) receptor play critical roles as mechanosensors in the regulation of heart contraction, growth and leading to heart failure. Integrin link the extracellular matrix and the intracellular cytoskeleton to initiate the mechanical signalling, whereas, the AT1 receptor could be activated by mechanical stress through an angiotensin-II-independent mechanism. Recent studies show that both Integrin and AT1 receptor and their downstream signalling factors including MAPKs, AKT, FAK, ILK and GTPase regulate heart function in cardiac myocytes. In this review we describe the role of mechanical sensors residing within the plasma membrane, mechanical sensor induced downstream signalling factors and its potential roles in cardiac contraction and growth.

Hepatic alterations are accompanied by changes to bile acid transporter-expressing neurons in the hypothalamus after traumatic brain injury

Annually, there are over 2 million incidents of traumatic brain injury (TBI) and treatment options are non-existent. While many TBI studies have focused on the brain, peripheral contributions involving the digestive and immune systems are emerging as factors involved in the various symptomology associated with TBI. We hypothesized that TBI would alter hepatic function, including bile acid system machinery in the liver and brain. The results show activation of the hepatic acute phase response by 2 hours after TBI, hepatic inflammation by 6 hours after TBI and a decrease in hepatic transcription factors, Gli 1, Gli 2, Gli 3 at 2 and 24 hrs after TBI. Bile acid receptors and transporters were decreased as early as 2 hrs after TBI until at least 24 hrs after TBI. Quantification of bile acid transporter, ASBT-expressing neurons in the hypothalamus, revealed a significant decrease following TBI. These results are the first to show such changes following a TBI, and are compatible with previous studies of the bile acid system in stroke models. The data support the emerging idea of a systemic influence to neurological disorders and point to the need for future studies to better define specific mechanisms of action.

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.