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Dementia is a complex syndrome with various presentations depending on the underlying pathologies. Low emission of transcranial near-infrared (tNIR) light can reach human brain parenchyma and be beneficial to a number of neurological and neurodegenerative disorders. We hereby examined the safety and potential therapeutic benefits of tNIR light stimulations in the treatment of dementia. Patients of mild to moderate dementia were randomized into active and sham treatment groups at 2:1 ratio. Active treatment consisted of low power tNIR light stimulations with an active photobiomodulation for 6 min twice daily during 8 consequent weeks. Sham treatment consisted of same treatment routine with a sham device. Neuropsychological battery was obtained before and after treatment. Analysis of variance (ANOVA) was used to analyze outcomes. Sixty subjects were enrolled. Fifty-seven subjects completed the study and had not reported health or adverse side effects during or after the treatment. Three subjects dropped out from trial for health issues unrelated to use of tNIR light treatment. Treatment with active device resulted in improvements of cognitive functions and changes were: an average increase of MMSE by 4.8 points; Logical Memory Tests I and II by ~3.0 points; Trail Making Tests A and B by ~24%; Boston Naming Test by ~9%; improvement of both Auditory Verbal Learning Tests in all subtest categories and overall time of performance. Many patients reported improved sleep after ~7 days of treatment. Caregivers noted that patients had less anxiety, improved mood, energy, and positive daily routine after ~14-21 days of treatment. The tNIR light treatments demonstrated safety and positive cognitive improvements in patients with dementia. Developed treatment protocol can be conveniently used at home. This study suggests that additional dementia treatment trials are warranted with a focus on mitigating caregivers' burden with tNIR light treatment of dementia patients.

Alzheimer’s disease (AD) is a neurodegenerative disease and the world’s primary cause of dementia, a condition characterized by significant progressive declines in memory and intellectual capacities. While dementia is the main symptom of Alzheimer’s, the disease presents with many other debilitating symptoms, and currently, there is no known treatment exists to stop its irreversible progression or cure the disease. Photobiomodulation has emerged as a very promising treatment for improving brain function, using light in the range from red to the near-infrared spectrum depending on the application, tissue penetration, and density of the target area. The goal of this comprehensive review is to discuss the most recent achievements in and mechanisms of AD pathogenesis with respect to neurodegeneration. It also provides an overview of the mechanisms of photobiomodulation associated with AD pathology and the benefits of transcranial near-infrared light treatment as a potential therapeutic solution. This review also discusses the older reports and hypotheses associated with the development of AD, as well as some other approved AD drugs.

Gulf War illness (GWI) is a chronic, multi-symptom disorder that has impacted approximately one third of Gulf War veterans. GWI and its symptoms have been linked to the exposure to neurological chemicals, including the anti-nerve gas drug pyridostigmine bromide (PB) and the insecticide permethrin (PER), among others. Mouse models utilizing these chemicals have reported symptomology analogous to human GWI. These changes include behavioral and cognitive impairment, neuroinflammation and hippocampal pathogenesis. Disease modifying interventions that target these pathological components are desperately needed. Vagus nerve stimulation (VNS) is FDA approved for drug-resistant epilepsy and depression. VNS has also been used off-label to target a myriad of symptoms, some of which are encompassed within the Kansas and CDC definitions of clinical GWI symptomology. A GWI animal model in which mice are exposed to a daily injection of PB and PER for 10 consecutive days has been shown to exhibit cognitive impairment and hippocampal pathology. The purpose of this study was to determine if 2-4 weeks of continuous vagus nerve stimulation initiated at 32 weeks after exposure to PB and PER would improve cognitive performance and hippocampal pathology. The results of the study revealed that exposure to PB and PER produces long-term cognitive deficits and reduced hippocampal neurogenesis. The results also showed that the VNS treatment was anxiolytic, improved some aspects of pattern separation deficits, and mitigated the reduced hippocampal neurogenesis. Thus, VNS improves outcomes in a mouse model of GWI and should be examined as a potential therapeutic strategy for mitigating some symptomology associated with GWI.

Background Transcranial near-infrared (tNIR) stimulation was proven to be a safe, reliable, and effective treatment for cognitive and behavioral symptoms of dementia. Dementia patients of different genders differ in terms of gross anatomy, biochemistry, genetic profile, clinical presentations, and socio-psychological status. Studies of the tNIR effect on dementia have thus far been gender-neutral, with dementia subjects being grouped based on diagnoses or dementia severity. This trial hereby investigated how dementia subjects of different sex respond to tNIR treatment. Methods A total of 60 patient-caregiver dyads were enrolled and randomized to this double-blind, sham-controlled clinical trial. The tNIR light has a wavelength of 1,060 nm to 1,080 nm and was delivered via a photobiomodulation (PBM) unit. The active PBM unit emits near-infrared (NIR) light while the sham unit does not. The treatment consists of a six-minute tNIR light stimulation session twice daily for eight weeks. Neuropsychological assessments conducted at baseline (week 0) and endline (week 8) were compared within the female and male group and between different sex, respectively. Results Over the course of treatment, active-arm female subjects had a 20.2% improvement in Mini‐Mental State Exam (MMSE) (mean 4.8 points increase, p < 0.001) and active-arm male cohort had 19.3% improvement (p < 0.001). Control-arm female subjects had a 6.5% improvement in MMSE (mean 1.5 points increase, p < 0.03) and control-arm male subjects had 5.9% improvement (p = 0.35) with no significant differences in the mean MMSE between female and male subjects in both arms respectively. Other comparison of assessments including Clock Copying and Drawing Test, Logical Memory Test - immediate and delayed recall yielded nominal but not statistically significant differences. No significant differences were observed in the mean MMSE between female and male subjects in both arms respectively before treatment implementation (active arm, p = 0.12; control arm, p = 0.50) at week 0, or after treatment completion (active arm, p = 0.11; control arm, p = 0.74) at week 8. Conclusion Despite differences between female and male dementia subjects, the response to tNIR light stimulation does not demonstrate gender-based differences. Further studies are warranted to refine the tNIR treatment protocol for subjects suffering from dementia or dementia-related symptoms.

Introduction: Primary central nervous system (CNS) tumors constitute a group of rare and heterogeneous tumors. A rising incidence rate in the United States has been linked to modern changes in early diagnosis and reporting. This study aims to examine temporal incidence trends, geographic variation, and the average annual age-adjusted rates among Hispanic populations in Texas from 1995 to 2013. Methods: SEER*STAT (Surveillance Research Program, National Cancer Institute (seer.cancer.gov/seerstat) version 8.3.2) and Joinpoint Regression 4.4.0.0 (Statistical Methodology and Applications Branch, Surveillance Research Program, National Cancer Institute) software were used to analyze incidence of primary brain and CNS tumors among Texas residents. Data were obtained from the Texas Cancer Registry of the Texas Department of State Health Services. Results: From the 30,122 cases of primary CNS tumors diagnosed throughout the state of Texas from 2008 to 2012, the overall average annual age-adjusted incidence rate for Hispanics and non-Hispanics combined was 25.35 per 100,000 persons. Among Hispanics, West Texas had the highest incidence trends and the highest average age-adjusted incidence rate of 27.17, followed by North Texas at 26.01 and the Panhandle at 23.63. East Texas had the lowest incidence rate of 16.23. The incidence trend among Hispanics has decreased consistently at a rate of 0.83 % from 1995 to 2013. Conclusions: The incidence of tumors was more pronounced in the Hispanic population in northern Texas compared with southern Texas. The presence of oil and gas production industries with farming and construction could play a role in the observed incidence of disease. Further studies with a focus on occupational health among Hispanics in Texas will be needed to elucidate the cause of such distribution.

Introduction The epidemiological factors surrounding astrocytoma and gliomas have been studied with little avail. Even limited conclusions have not been reached in spite of significant past research efforts. Ionizing radiation is currently one of the only factors consistently associated with glioma formation. Studies in an attempt to link environmental and occupational exposures with brain neoplasms have continued to produce inconsistent results. This study aims to explore the distribution and epidemiology of astrocytomas within a Central Texas patient population in order to elucidate any possible differences in epidemiologic and prognostic factors based on race, histology, and primary tumor site. Methods Eight hundred forty-five clinical cases with the diagnosis of astrocytoma were retrospectively obtained from the tumor registry of the Scott & White Integrated Healthcare System from 1976 to 2014. We investigated the effects of gender, race, tumor histology, tumor site, treatment methods, and mortality of this cohort of patients in Central Texas. Results Prevalence data echoes that of the national epidemiology in that among our sample, White individuals had the highest prevalence (n=666, 78.8%), followed by Hispanics (n=94, 11.1%) and Black individuals (n=78, 9.2%). White patients had higher rates of parietal lobe (6.6% vs. 0.6%, p

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor with dismal survival. This study aims to examine the prognostic value of primary tumor sites and race on survival outcomes.Patient data obtained from the Scott and White Hospital Brain Tumor Registry (1976-2013) were stratified according to sex, age, race, primary tumor site, vital status, and survival.Of the 645 patients, 580 (89.9%) were diagnosed with GBM not otherwise specified (GBM NOS), 57 (8.8%) with GBM, and 8 (1.2%) with giant-cell GBM. Most were male (53.5%), aged 50 years or older (78.7%). The white population had the highest GBM prevalence (87.1%) and the lowest overall survival versus all other race groups (6.6% vs. 30.1%; P0.01). The black population had a relatively low prevalence of GBM (5.9%) and the greatest overall survival versus all others (47.4% vs. 7.3%; P0.01). Primary tumor sites located in the temporal (25.8% vs. 20.2%; P = 0.03), occipital (8.1% vs. 2.9%; P = 0.05), and parietal lobes (24.2% vs. 20.8%; P = 0.05) had a greater occurrence in surviving individuals. The overall survival for men versus women was (62.9% vs. 37.1%; P = 0.12).Black racial background and temporal, occipital, or parietal primary tumor sites are suggestive of positive survival outcomes. Conversely, white racial background with primary tumor sites in the brain overlapping and NOS areas seem to be associated with negative outcomes and decreased survival. Thus, racial background and primary tumor site may be useful prognostic factors in patients with GBM.

Background Although rare, primary central nervous system (CNS) tumors are associated with significant morbidity and mortality. Texas is a representative sample of the United States population given its large population, ethnic disparities, geographic variations, and socio-economic differences. This study used Texas data to determine if variations in incidence trends and rates exist among different ethnicities in Texas. Methods Data from the Texas Cancer Registry from 1995 to 2013 were examined. Joinpoint Regression Program software was used to obtain the incidence trends and SEER*Stat software was used to produce average annual age-adjusted incidence rates for both nonmalignant and malignant tumors in Texas from 2009 to 2013. Results The incidence trend of malignant primary CNS tumors in whites was stable from 1995 to 2002, after which the annual percent change decreased by 0.99% through 2013 (95% CI, -1.4, -0.5; P = .04). Blacks and Asian/Pacific Islanders showed unchanged incidence trends from 1995 to 2013. Hispanics had an annual percent change of -0.83 (95% CI, -1.4, -0.2; P = .009) per year from 1995 through 2013. From 2009 to 2013, the incidence rates of nonmalignant and malignant primary CNS tumors were highest among blacks, followed by whites, Hispanics, Asians, and American Indians/Alaskan Natives. Conclusions Consistent with the 2016 Central Brain Tumor Registry of the United States report, the black population in Texas showed the highest total incidence of CNS tumors of any other race studied. Many factors have been proposed to account for the observed differences in incidence rate including geography, socioeconomic factors, and poverty factors, although the evidence for these external factors is lacking.

We describe the combination of photobiomodulation using the Cognitolite helmet with 1068 nm light emitting diodes (LEDs) with neurofeedback and quantitative electroencephalogram measurements to treat humans with cognitive impairment and dementia. The most significant changes in transcortical electrical activity were the normalization of central alpha (8–12 Hz) amplitudes and delta (0–4 Hz) and theta (4–8 Hz) hypocoherence and phase lag. Patients receiving real PBM improved on various cognitive tests including the mini mental state exam, clock drawing, and memory tests.

Gulf war illness (GWI) is a chronic multi-symptom disease that afflicts 25-33% of troops that were deployed in the 1990-1991 Gulf War. GWI symptoms include cognitive, behavioral and emotional deficits, as well as migraines and pain. It is possible that exposure to Gulf War agents and prophylactics contributed to the reported symptomology. Pyridostigmine bromide (PB) and permethrin (PER) were given to protect from nerve gas attacks and insect vector born disease, respectively. Previous studies have demonstrated that 10 days of exposure to these chemicals can cause symptoms analogous to those observed in GWI, including impairment of long-term memory in mice. Other studies using this model have shown chronic neuroinflammation, and chronic neuroinflammation can lead to altered nociceptive sensitivity. At 10-weeks after the 10-day PB and PER exposure paradigm, we observed lowered nociceptive threshold on the Von Frey test that was no longer evident at 28 weeks and 38 weeks post-exposure. We further determined that vagus nerve stimulation, initiated at 38 weeks after exposure, restores the lowered nociceptive sensitivity. Therefore, stimulating the vagus nerve appears to influence nociception. Future studies are need to elucidate possible mechanisms of this effect.

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.

Purpose of review The purpose of this review was to discuss therapeutic manipulations and effective current interventions available to treat intravascular lymphoma in the central nervous system. Recent findings Patients experienced resolution and remission of disease for 14 months and up to 2 years after eight cycles of R-CHOP and four courses of intrathecal therapy with MTX, cytarabine, and prednisolone. Intravascular use of unfractionated heparin during therapy may contribute to better outcome. Summary Series of therapeutic avenues were analyzed and compared. The effective current treatment of intravascular lymphoma in the CNS is considered to be a combinational intrathecal methotrexate-based chemotherapy with rituximab. Since intrathecal administration bypasses the blood-brain barrier, lower doses can be given, which thereby minimizes systemic toxicity. Practical use of intrathecal chemotherapy is also justified for prophylaxis in intravascular lymphoma-diagnosed patients without CNS involvement.

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 (2–30min), whereas marked activation of ERK1/2 and p38 occurred much later (2–4h). β 1D -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 β 1 -integrin and the differential actions of MAP kinase family members.

Today, Lyme disease is the most commonly reported tick-borne disease in the United States and Europe. The culprits behind Lyme disease are the Borrelia species of bacteria. In the USA, Borrelia burgdorferi causes the majority of cases, while in Europe and Asia Borrelia afzelii and Borrelia garinii carry the greatest burden of disease. The clinical manifestations of Lyme disease have been identified as early localized, early disseminated, and late chronic. The neurological effects of Lyme disease include both peripheral and central nervous systems involvement, including focal nerve abnormalities, cranial neuropathies, painful radiculoneuritis, meningitis, and/or toxic metabolic encephalopathy, known as Lyme encephalopathy. Given the geographic predominance of Lyme disease in the Northeast and Midwest of the USA, no major studies have been conducted regarding Southern states. Between 2005 and 2014, the Center for Disease Control has reported 582 confirmed cases of Lyme disease in Texas. Because of the potential for increased incidence and prevalence in Texas, it has become essential for research and clinical efforts to be diverted to the region. The Texas A&M College of Veterinary Medicine and Biomedical Sciences Lyme Lab has been investigating the ecology of Lyme disease in Texas and developing a pan-specific serological test for Lyme diagnosis. This report aimed to exposure materials and raise awareness of Lyme disease to healthcare providers.

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 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.

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.

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.

Mechanical stretch is a major factor for myocardial hypertrophy and heart failure. Stretch activates mechanical sensors from cardiac myocytes, leading to a series of signal transduction cascades, which can result in cell malfunction and remodeling. It is well known that mechanical stretch also induces the release of paracrine factors from cardiac fibroblasts, as well as myocytes. Due to complicated circumstance of heart tissue, it is difficult to fully investigate the characteristics of these factors in situ. Here we describe static stretch and conditioned medium experiments as methods to examine the function of paracrine factors between primary cultured cardiac myocytes and fibroblasts.

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.

Mechanical stretch is a major determinant that leads to heart failure, which is associated with a steady increase in myocardial angiotensinogen (Aogen) expression and formation of the biological peptide angiotensin II (Ang II). c-jun NH2-terminal kinase (JNK) and p38α have been found to have opposing roles on stretch-induced Aogen gene expression in neonatal rat ventricular myocytes (NRVM). JNK negatively regulated Aogen expression in NRVM following acute stretch, whereas with prolonged stretch, JNK phosphorylation was downregulated and p38α was found responsible for upregulation of Aogen expression. However, the mechanisms responsible for regulation of these kinases, especially the cross-talk between p38 and JNK1/2, remain to be determined. In this study, a combination of pharmacologic and molecular approaches (adenovirus-mediated gene transfer) were used to examine the mechanisms by which p38 regulates JNK phosphorylation in NRVM under stretch and non-stretch conditions. Pharmacologic inhibition of p38 significantly increased JNK phosphorylation in NRVM at 15 min, whereas overexpression of wild-type p38α significantly decreased JNK phosphorylation. While p38α overexpression prevented stretch-induced JNK phosphorylation, pharmacologic p38 inhibition abolished the JNK dephosphorylation during 15-60 min of stretch. Expression of constitutively-active MKK3 (MKK3CA), the upstream activator of p38, abolished JNK phosphorylation in both basal and stretched NRVM. Pharmacologic inhibition of MAP kinase phosphatase-1 (MKP-1) or protein phosphatase-1 (PP1) increased JNK phosphorylation in NRVM, suggesting the involvement of these phosphatases on reversing stretch-induced JNK activation. Inhibition of MKP-1, but not PP1, reduced JNK phosphorylation in NRVM overexpressing MKK3CA under basal conditions (no-stretch). Inhibition of MKP-1 also enhanced stretch-induced JNK phosphorylation in NRVM at 15 to 60 min. In summary, these results indicate that MKP-1 inhibits JNK phosphorylation in stretched NRVM through p38 dependent and independent mechanisms, whereas PP1 regulates JNK through a p38-independent mechanism.

Mechanical stretch is a major factor for myocardial hypertrophy and heart failure. Stretch activates mechanical sensors from cardiac myocytes, leading to a series of signal transduction cascades, which can result in cell malfunction and remodeling. It is well known that mechanical stretch also induces the release of paracrine factors from cardiac fibroblasts, as well as myocytes. Due to complicated circumstance of heart tissue, it is difficult to fully investigate the characteristics of these factors in situ. Here we describe static stretch and conditioned medium experiments as methods to examine the function of paracrine factors between primary cultured cardiac myocytes and fibroblasts.

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.

Recent molecular and pharmacological studies performed on non-cardiac tissues suggest that tumor endothelial marker-8 (TEM-8), also known as anthrax toxin receptor 1 (ANTXR1) and capillary morphogenesis gene-2 (CMG-2), known as anthrax toxin receptor 2 (ANTXR2) regulate survival, proliferation, cell shape, as well as polarity, adhesion, migration and differentiation. However, downstream signaling mechanisms and function of these molecules have not been studied. Recent studies indicate that activation of protein kinase B (Akt) and c-Jun N-terminal kinases (JNK) have been associated with hypertrophic growth, as well as regulation of contractility in cardiac myocytes (CM). In this study we’ve investigated the role of ANTXR1 and ANTXR2 receptors in intracellular pathways of contractility regulation in neonatal rat ventricular myocytes (NRVM). Primary culture of NRVM was used to determine the effects of ANTXR1/ANTXR2 activation on Akt and JNK phosphorylation by Western blot analysis. Flow cytometry and immunostaining of alive CM were used to determine expression levels and distribution of anthrax receptors throughout the cells. Furin-activated form of anthrax protective antigen (PA 63 ) was used in order to selectively stimulate ANTXR1/ANTXR2 receptors and get understanding of downstream signaling mechanisms coupled to them. The treatments with angiotensin II, type1 receptor (AT 1 ) inhibitors were used in order to separate responses of distinct regulatory pathways of contractility. All treatments were done at variable time courses started in seconds and finished in half of hour to determine a sequence of phosphorylation reactions going from cellular membrane to deep inside of cell. As a result, abundant expression of both receptors was observed in cardiac myocytes. Activation of one phospho-site of focal adhesion kinase (FAK 861 ) was started in 15 sec with subsequent phosphorylation of JNK and Akt 473 in 5 and 10 minutes. Involvement of new molecules was observed in cascade chain reaction of contractility regulation. In conclusion, ANTXR1/ANTXR2 were demonstrated to couple to mechano-sensor molecules: FAK, Akt and JNK, as well as interact with the AT 1 receptors to mediate downstream signaling events responsible for regulation of contractility.

Cardiac hypertrophy is a common response to mechanical stimuli, such as increased hemodynamic load and exercise. However, the underlying mechanotransduction processes are poorly understood. Recent studies indicate that both β 1 integrin and angiotensin type I receptor (AT 1 R) can serve as mechanoreceptors in the intact myocardium and isolated cardiac myocytes. Mechanical stress induced activation of Akt and JNK has been associated with various aspects of hypertrophic growth. We used primary cultures of NRVM plated on deformable membranes to test whether static stretch-induced activation (phosphorylation) of Akt and JNK involves β 1 integrin and/or AT 1 R. In these studies, pretreatment with the AT 1 R antagonist Losartan (10 µM), significantly decreased mechanical stretch induced activation of Akt (Akt 473 , 47.1 ± 6.1%, p308 , 33.1 ± 3.2%, p1 D -integrin (Tac-β 1 D ) alone had no effect on stretch-induced Akt and JNK activation, the addition of Losartan to the culture medium decreased stretch-induced activation of both Akt (Akt 473 , 74.9 ± 10.4%, p308 , 66.1 ± 8%, p473 , 36.3 ± 7.6%, p308 , 29.8 ± 6.2%, p473 , 64.9 ± 10.4%, p308 , 50.8 ± 7.8%, p1 R, whereas JNK activation could be mediated through either AT 1 R or β 1 integrin. The regulation Akt and JNK was also found to be significantly dependent on Rac1 activation. In conclusion, this study suggests that both AT 1 R and β 1 integrin are important mechanosensors that activate pathways that lead to cardiac myocyte hypertrophy.

We previously demonstrated that anthrax lethal toxin (LT, protective antigen [PA] + lethal factor [LF]) produces acute systolic heart failure, characterized by increased left ventricular (LV) end-diastolic volume, reduced ejection fraction and decreased contractility in male Sprague-Dawley (SD) rats. The molecular basis for the cardiac dysfunction observed in either an animal model or clinical setting of anthrax toxicity remains to be examined. Previously, we tested LT dose-response effects on isolated neonatal rat ventricular myocytes (NRVM) at a dose of 0.05 ng/mL PA + 0.025 ng/mL LF diminished PLB and JNK phosphorylation from 1h to 4 h. During the LT+PA time-course, Akt phosphorylation at both Thr 308 and Ser 473 were also significantly reduced at all time points from 30 min to 4 h, accompanied by elevated B56α total protein and intracellular Ca 2+ , and reduced PLB phosphorylation. This response is mediated by translocation of LF into the cell through the anthrax receptors 1 and 2 (ATR1/2), which are structurally similar to integrins. In this study, we tested whether stimulation of ATR1/2 by PA alone can induce activation of the integrin effectors, FAK and JNK. Cardiac myocytes were treated time dependently (5 sec - 30 min) by PA at 0.25 ng/mL, and activation of FAK protein at different residues was determined. PA induced phosphorylation of FAK-Tyr 861 at 15 sec (p397 or FAK-Tyr 925 phosphorylation for any of the time points. Furthermore, we found that PA alone maximally increased JNK phosphorylation at 15 min of treatment (pB. anthracis PA stimulation and involvement in acute responses in regulation of FAK and JNK activation in independent of AT1 and EGFR pathway.

Mechanical stretch is a major determinant that leads to heart failure. Heart failure is associated with a steady increase in myocardial angiotensinogen (Ao) expression and the biological peptide angiotensin II (Ang II) formation. We have previously identified key downstream effectors that couple to mechanical stretch-induced Ao gene expression. JNK1/2 and p38α were found to have opposing roles on stretch-induced Ao gene expression in neonatal rat cardiac myocytes (NRVM) and cardiac fibroblasts (NRFB). JNK negatively regulated Ao expression in NRVM and NRFB following acute stretch, whereas with prolonged stretch, p38α was responsible for upregulation of Ao expression. However, the mechanisms leading to activation of these kinases and cross-talk between these signal molecules remain to be determined. Epidermal growth factor receptor (EGFR) is expressed in both NRVM and NRFB, but its precise role in mechanosensing is poorly understood. In NRVM, stretch caused JNK1/2 activation (phosphorylation) within 2 min, with a peak response at 15 min (3.96 ± 0.71 fold, p

Background: Several Helicobacter pylori proteins have been reported to be associated with severe symptoms of gastric disease. However, expression levels of most of these disease-associated proteins require further evaluation in order to clarify their relationships with gastric disease patterns. Representative proteome components of 71 clinical isolates of H. pylori were analyzed quantitatively to determine whether the protein expression levels were associated with gastric diseases and to cluster clinical isolates. Methods: After two-dimensional electrophoresis (2-DE) of H. pylori isolates, spot intensities were analyzed using pdquest 2-D Gel Analysis Software. The intensities of 10 representative protein spots, identified by peptide fingerprinting using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) or peptide sequencing using quadrupole TOF MS, were subjected to the nonparametric Mann–Whitney test and hierarchical agglomerative cluster analysis. The relationship between clusters and gastric diseases was analyzed by the chi-squared test. Results: Although the spot intensities of the 10 representative proteins were highly variable within each gastric disease group, the expression levels of CagA, UreB, GroEL, EF-Tu, EF-P, TagD, and FldA showed some significant differences among the gastric disease patterns. On the basis of the 10 target protein intensities, hierarchical agglomerative cluster analysis generated a dendrogram with clusters indicative of chronic gastritis/gastric cancers and gastric/duodenal ulcers. Conclusion: These results indicated that quantitative analysis of proteome components is a feasible method for examining disease-associated proteins and clustering clinical strains of H. pylori.