Chhanda Bose

Nicotine-induced proliferation of isolated rat pancreatic acinar cells: effect on cell signalling and function.

Abstract.  Objectives: The aim of the current study was to investigate whether nicotine treatment would induce the proliferation of isolated rat primary pancreatic acinar cells in culture by activating mitogen‐activated protein kinase (MAPK) signalling and exocrine secretion. Materials and Methods: A nicotine dose‐ and time‐response curve was initially developed to determine the optimal dose and time used for all subsequent studies. Proliferation studies were conducted by cell counting and confirmed further by bromodeoxyuridine (BrdU) incorporation and flow cytometry assays. MAPK signalling studies were conducted by Western blot analysis. Localization of ERK1/2 signals, with or without nicotine and the MAPK inhibitor, was visualized by immunofluorescence. Results: Nicotine treatment caused dose‐dependent activation of extracellular signal‐regulated kinases (ERK1/2), the maxima occurring at 100 µm and at 3 min after treatment; the response was suppressed by the ERK1/2 inhibitor. Maximal nicotine‐induced cell proliferation occurred at 24 h, and UO126‐treatment significantly reduced this response. Exposure of cells to 100 µm nicotine for 6 min significantly enhanced both baseline and cholecystokinin‐stimulated cell function, and these effects were not affected by treatment with the inhibitor of ERK1/2 but were suppressed by mecamylamine, a nicotinic receptor antagonist. Conclusions: Our results suggest that nicotine treatment induced cell proliferation of isolated pancreatic acinar cells and that this is coupled with the activation of MAPK signalling with no effect on its function. Hence, in primary cells, the mechanism of induction and regulation of these two processes, cell proliferation and cell function, by nicotine treatment are independent of each other.

Age-related alteration in hepatic acyl-CoA : cholesterol acyltransferase and its relation to LDL receptor and MAPK

The aim of this study was to evaluate changes in the regulation of lipid metabolism and mitogen-activated protein kinases (MAPK) in the liver of C57BL/6 mice as they age. This was done by assessing the status of total cholesterol content and its enzyme, acyl-CoA: cholesterol acyltransferase (ACAT), in liver microsomal preparations and the low-density lipoprotein receptor (LDLr) mRNA expression in the livers of 4-24-month-old C57B/6 mice, without exogenous cholesterol feeding. With aging, there was an increase in cholesterol content and ACAT activity in liver microsomes. Northern blot analysis and real-time quantitative polymerase chain reaction data showed that ACAT-2 mRNA increased with age as well. LDLr expression decreased significantly in an age-dependent manner. In addition, we studied the basal and activated forms of MAPK, e.g. extracellular regulatory kinase (ERK-1/2), c-jun NH2-terminal kinase (JNK-1/2) and p38 MAPK. During aging, there was a considerable decrease in phosphorylated ERK-1/2 level while JNK-1/2 and p38 MAPK levels increased with age. Our studies showed an altered LDLr expression and altered phosphorylated MAPK in the liver of C57BL/6 mice during aging. These alterations might contribute to the development of atherosclerosis, hypercholesterolemia and other cholesterol-related conditions.

Gsta4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity *

The alpha class glutathione s-transferase (GST) isozyme GSTA4-4 (EC2.5.1.18) exhibits high catalytic efficiency to-wards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated concentrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To examine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we disrupted the Gsta4 gene that encodes GSTA4-4 in mice. The effect of electrophile and oxidants on embryonic fibroblasts (MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of GSTA4-4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)3], a pro-drug form of 4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of caspase-3. Interestingly, when recombinant mammalian or invertebrate GSTs were delivered to Gsta4 null MEF, activation of stress-related kinases in 4-HNE(Ac)3 treated Gsta4 null MEF were inversely correlated with the catalytic efficiency of delivered GSTs towards 4-HNE. Our data suggest that GSTA4-4 plays a major role in protecting cells from the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE.

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH(2) terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells.

Gadolinium Contrast Agent-Induced CD163+ Ferroportin+ Osteogenic Cells in Nephrogenic Systemic Fibrosis

Gadolinium-based contrast agents are linked to nephrogenic systemic fibrosis in patients with renal insufficiency. The pathology of nephrogenic systemic fibrosis is characterized by abnormal tissue repair: fibrosis and ectopic ossification. The mechanisms by which gadolinium could induce fibrosis and ossification are not known. We examined in vitro the effect of a gadolinium-based contrast agent on human peripheral blood mononuclear cells for phenotype and function relevant to the pathology of nephrogenic systemic fibrosis using immunofluorescence, flow cytometry, real-time PCR, and osteogenic assays. We also examined tissues from patients with nephrogenic systemic fibrosis, using IHC to identify the presence of cells with phenotype induced by gadolinium. Gadolinium contrast induced differentiation of human peripheral blood mononuclear cells into a unique cellular phenotype--CD163(+) cells expressing proteins involved in fibrosis and bone formation. These cells express fibroblast growth factor (FGF)23, osteoblast transcription factors Runt-related transcription factor 2, and osterix, and show an osteogenic phenotype in in vitro assays. We show in vivo the presence of CD163(+)/procollagen-1(+)/osteocalcin(+) cells in the fibrotic and calcified tissues of nephrogenic systemic fibrosis patients. Gadolinium contrast-induced CD163(+)/ferroportin(+)/FGF23(+) cells with osteogenic potential may play a role in systemic fibrosis and ectopic ossification in nephrogenic systemic fibrosis.

Carbamylated Low-Density Lipoprotein (cLDL)-Mediated Induction of Autophagy and Its Role in Endothelial Cell Injury.

Patients with chronic kidney disease (CKD) have high risk of cardiovascular complications. Plasma levels of carbamylated proteins produced by urea-derived isocyanate or thiocyanate are elevated in CKD patients and that they are significant predictors of cardiovascular events and all-cause mortality. Carbamylated LDL (cLDL) has pro-atherogenic properties and is known to affect major biological processes relevant to atherosclerosis including endothelial cell injury. The underlying mechanisms of cLDL-induced endothelial cell injury are not well understood. Although autophagy has been implicated in atherosclerosis, cLDL-mediated induction of autophagy and its role in endothelial cell injury is unknown. Our studies demonstrate that human coronary artery endothelial cells (HCAECs) respond to cLDL by specific induction of key autophagy proteins including LC3-I, beclin-1, Atg5, formation of lipid-conjugated LC3-II protein, and formation of punctate dots of autophagosome-associated LC3-II. We demonstrated that autophagy induction is an immediate response to cLDL and occurred in a dose and time-dependent manner. Inhibition of cLDL-induced autophagy by a specific siRNA to LC3 as well as by an autophagy inhibitor provided protection from cLDL-induced cell death and DNA fragmentation. Our studies demonstrate that autophagy plays an important role in cLDL-mediated endothelial cell injury and may provide one of the underlying mechanisms for the pathogenesis of cLDL-induced atherosclerosis in CKD patients.

Recent Advances in Understanding the Pathogenesis of Atherosclerosis in CKD Patients

A need exists for developing new therapies to improve cardiovascular outcomes in end-stage kidney disease. Three new areas that address novel pathophysiological mechanisms and/or therapeutic approaches toward cardiovascular events in chronic kidney disease patients include the use of an anti-inflammatory agent, the role of catalytic iron, and protein carbamylation. In preliminary studies, hydroxychloroquine, which has multiple anti-inflammatory properties, preserved vascular compliance for the aorta and major vessels, as well as reduced the extent of severity of atherosclerosis in ApoE-/- mice. The ability of iron to rapidly and reversibly cycle between 2 oxidation states makes iron potentially hazardous by enabling it to participate in the generation of powerful oxidant species. We have shown that high catalytic iron in the general population is associated with a 4-fold increase in prevalent cardiovascular disease (CVD), even after accounting for traditional risk factors. In addition, the highest levels of catalytic iron are present in dialysis patients and, more specifically, patients with prevalent CVD have several-fold higher catalytic iron levels compared with controls without CVD. These data suggest the utility of iron chelators for preventing and treating CVD in patients with chronic kidney disease and should be further investigated. Carbamylation of proteins results from nonenzymatic chemical modification by isocyanic acid derived from urea and an alternative route, the myeloperoxidase-catalyzed oxidation of thiocyanate. We have shown carbamylated low-density lipoprotein to have all the major biological effects relevant to atherosclerosis including endothelial cell injury, increased expression of cell adhesion molecules, and vascular smooth muscle cell proliferation. In 2 separate clinical studies, plasma levels of carbamylated protein independently predicted an increased risk of CVD and death.

Evidence Suggesting a Role of Iron in a Mouse Model of Nephrogenic Systemic Fibrosis

Nephrogenic systemic fibrosis is associated with gadolinium contrast exposure in patients with reduced kidney function and carries high morbidity and mortality. We have previously demonstrated that gadolinium contrast agents induce in vivo systemic iron mobilization and in vitro differentiation of peripheral blood mononuclear cells into ferroportin (iron exporter)-expressing fibrocytic cells. In the present study we examined the role of iron in a mouse model of nephrogenic systemic fibrosis. Chronic kidney disease was induced in 8-week-old male Balb/C mice with a two-step 5/6 nephrectomy surgery. Five groups of mice were studied: control (n = 5), sham surgery control (n = 5), chronic kidney disease control (n = 4), chronic kidney disease injected with 0.5 mmol/kg body weight of Omniscan 3 days per week, for a total of 10 injections (n = 8), and chronic kidney disease with Omniscan plus deferiprone, 125 mg/kg, in drinking water (n = 9). Deferiprone was continued for 16 weeks until the end of the experiment. Mice with chronic kidney disease injected with Omniscan developed skin changes characteristic of nephrogenic systemic fibrosis including hair loss, reddening, ulceration, and skin tightening by 10 to 16 weeks. Histopathological sections demonstrated dermal fibrosis with increased skin thickness (0.25±0.06 mm, sham; 0.34±+0.3 mm, Omniscan-injected). Additionally, we observed an increase in tissue infiltration of ferroportin-expressing, fibrocyte-like cells accompanied by tissue iron accumulation in the skin of the Omniscan-treated mice. The deferiprone-treated group had significantly decreased skin thickness (p<0.05) and significantly decreased dermal fibrosis compared to the Omniscan-only group. In addition, iron chelation prevented tissue infiltration of ferroportin-expressing, fibrocyte-like cells. Our in vitro experiments demonstrated that exposure to Omniscan resulted in the release of catalytic iron and this was prevented by the iron chelator deferiprone. Deferiprone inhibited the differentiation of human peripheral blood mononuclear cells into ferroportin-expressing cells by immunohistochemical staining and western blot analysis. Our studies support an important role of iron in the pathophysiology of gadolinium chelate toxicity and nephrogenic systemic fibrosis.

Impact of Hydroxychloroquine on Atherosclerosis and Vascular Stiffness in the Presence of Chronic Kidney Disease.

Cardiovascular disease is the largest cause of morbidity and mortality among patients with chronic kidney disease (CKD) and end-stage kidney disease, with nearly half of all deaths attributed to cardiovascular disease. Hydroxychloroquine (HCQ), an anti-inflammatory drug, has been shown to have multiple pleiotropic actions relevant to atherosclerosis. We conducted a proof-of-efficacy study to evaluate the effects of hydroxychloroquine in an animal model of atherosclerosis in ApoE knockout mice with and without chronic kidney disease. Forty male, 6-week-old mice were divided into four groups in a 2 x 2 design: sham placebo group; sham treatment group; CKD placebo group; and CKD treatment group. CKD was induced by a two-step surgical procedure. All mice received a high-fat diet through the study duration and were sacrificed after 16 weeks of therapy. Mice were monitored with ante-mortem ultrasonic echography (AUE) for atherosclerosis and vascular stiffness and with post-mortem histology studies for atherosclerosis. Therapy with HCQ significantly reduced the severity of atherosclerosis in CKD mice and sham treated mice. HCQ reduced the area of aortic atherosclerosis on en face examination by approximately 60% in HCQ treated groups compared to the non-treated groups. Additionally, therapy with HCQ resulted in significant reduction in vascular endothelial dysfunction with improvement in vascular elasticity and flow patterns and better-preserved vascular wall thickness across multiple vascular beds. More importantly, we found that presence of CKD had no mitigating effect on HCQ’s anti-atherosclerotic and vasculoprotective effects. These beneficial effects were not due to any significant effect of HCQ on inflammation, renal function, or lipid profile at the end of 16 weeks of therapy. This study, which demonstrates structural and functional protection against atherosclerosis by HCQ, provides a rationale to evaluate its use in CKD patients. Further studies are needed to define the exact mechanisms through which HCQ confers these benefits.

Sulforaphane potentiates anticancer effects of doxorubicin and attenuates its cardiotoxicity in a breast cancer model

Breast cancer is the most common malignancy in women of the Western world. Doxorubicin (DOX) continues to be used extensively to treat early-stage or node-positive breast cancer, human epidermal growth factor receptor-2 (HER2)-positive breast cancer, and metastatic disease. We have previously demonstrated in a mouse model that sulforaphane (SFN), an isothiocyanate isolated from cruciferous vegetables, protects the heart from DOX-induced toxicity and damage. However, the effects of SFN on the chemotherapeutic efficacy of DOX in breast cancer are not known. Present studies were designed to investigate whether SFN alters the effects of DOX on breast cancer regression while also acting as a cardioprotective agent. Studies on rat neonatal cardiomyocytes and multiple rat and human breast cancer cell lines revealed that SFN protects cardiac cells but not cancer cells from DOX toxicity. Results of studies in a rat orthotopic breast cancer model indicated that SFN enhanced the efficacy of DOX in regression of tumor growth, and that the DOX dosage required to treat the tumor could be reduced when SFN was administered concomitantly. Additionally, SFN enhanced mitochondrial respiration in the hearts of DOX-treated rats and reduced cardiac oxidative stress caused by DOX, as evidenced by the inhibition of lipid peroxidation, the activation of NF-E2-related factor 2 (Nrf2) and associated antioxidant enzymes. These studies indicate that SFN not only acts synergistically with DOX in cancer regression, but also protects the heart from DOX toxicity through Nrf2 activation and protection of mitochondrial integrity and functions.