Rajprasad Loganathan

CXCL10-induced cell death in neurons: role of calcium dysregulation

Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over‐expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimers disease and HIV‐associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10‐mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca2+ dysregulation in CXCL10‐mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca2+ and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca2+ elevations with the caspases that are involved in CXC10‐induced neuronal apoptosis. Our data showed that increased Ca2+, which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase‐9. This initiator caspase sequentially activates the effector caspase‐3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10‐mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up‐regulation.

Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are altered with type 1 diabetes due to the direct effects of hyperglycemia

BackgroundDiminished calcium (Ca2+) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. However, the mechanism responsible was unclear.Methodology/Principal FindingsVSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP3R) and the SR Ca2+ pumps (SERCA 2 and 3). Generally, a decrease in IP3R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP3R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca2+ in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca2+ sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca2+ in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca2+ in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca2+ responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.Conclusions/SignificanceThis work demonstrates that the previously-reported reduced Ca2+ signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP3R Ca2+ channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.

Cardiac dysfunction in the diabetic rat: quantitative evaluation using high resolution magnetic resonance imaging

BackgroundDiabetes is a major risk factor for cardiovascular disease. In particular, type 1 diabetes compromises the cardiac function of individuals at a relatively early age due to the protracted course of abnormal glucose homeostasis. The functional abnormalities of diabetic myocardium have been attributed to the pathological changes of diabetic cardiomyopathy.MethodsIn this study, we used high field magnetic resonance imaging (MRI) to evaluate the left ventricular functional characteristics of streptozotocin treated diabetic Sprague-Dawley rats (8 weeks disease duration) in comparison with age/sex matched controls.ResultsOur analyses of EKG gated cardiac MRI scans of the left ventricle showed a 28% decrease in the end-diastolic volume and 10% increase in the end-systolic volume of diabetic hearts compared to controls. Mean stroke volume and ejection fraction in diabetic rats were decreased (48% and 28%, respectively) compared to controls. Further, dV/dt changes were suggestive of phase sensitive differences in left ventricular kinetics across the cardiac cycle between diabetic and control rats.ConclusionThus, the MRI analyses of diabetic left ventricle suggest impairment of diastolic and systolic hemodynamics in this rat model of diabetic cardiomyopathy. Our studies also show that in vivo MRI could be used in the evaluation of cardiac dysfunction in this rat model of type 1 diabetes.

Electrocardiographic changes with the onset of diabetes and the impact of aerobic exercise training in the Zucker Diabetic Fatty (ZDF) rat

BackgroundEarly markers of diabetic autonomic neuropathy (DAN) in an electrocardiogram (ECG) include elevated R wave amplitudes, widening of QTc intervals and decreased heart rate variability (HRV). The severity of DAN has a direct relationship with mortality risk. Aerobic exercise training is a common recommendation for the delay and possible reversal of cardiac dysfunction. Limited research exists on ECG measures for the evaluation of aerobic exercise training in Zucker Diabetic Fatty (ZDF) rat, a model of type 2 diabetes. The objective of this study was to assess whether aerobic exercise training may attenuate diabetes induced ECG changes.MethodsMale ZDF (obese fa/fa) and control Zucker (lean fa/+) rats were assigned to 4 groups: sedentary control (SC), sedentary diabetic (SD), exercised control (EC) and exercised diabetic (ED). The exercised groups began 7 weeks of treadmill training after the development of diabetes in the ED group. Baseline (prior to the training) and termination measurements included body weight, heart weight, blood glucose and glycated hemoglobin levels and ECG parameters. One way repeated measures ANOVA (group) analyzed within and between subject differences and interactions. Pearson coefficients and descriptive statistics described variable relationships and animal characteristics.ResultsDiabetes caused crucial changes in R wave amplitudes (p < 0.001), heart rate variability (p < 0.01), QT intervals (p < 0.001) and QTc intervals (p < 0.001). R wave amplitude augmentation in SD rats from baseline to termination was ameliorated by exercise, resulting in R wave amplitude changes in ED animals similar to control rats. Aerobic exercise training neither attenuated QT or QTc interval prolongation nor restored decreases in HRV in diabetic rats.ConclusionThis study revealed alterations in R wave amplitudes, HRV, QT and QTc intervals in ZDF rats. Of these changes, aerobic exercise training was able to correct R wave amplitude changes. In addition, exercise has beneficial effect in this diabetic rat model in regards to ECG correlates of left ventricular mass.

Exercise-induced cardiac performance in autoimmune (Type 1) diabetes is associated with a decrease in myocardial diacylglycerol

One of the fundamental biochemical defects underlying the complications of diabetic cardiovascular system is elevation of diacylglycerol (DAG) and its effects on protein kinase C (PKC) signaling. It has been noted that exercise training attenuates poor cardiac performance in Type 1 diabetes. However, the role of PKC signaling in exercise-induced alleviation of cardiac abnormalities in diabetes is not clear. We investigated the possibility that exercise training modulates PKC-βII signaling to elicit its beneficial effects on the diabetic heart. bio-breeding diabetic resistant rats, a model reminiscent of Type 1 diabetes in humans, were randomly assigned to four groups: 1) nonexercised nondiabetic (NN); 2) nonexercised diabetic (ND); 3) exercised nondiabetic; and 4) exercised diabetic. Treadmill training was initiated upon the onset of diabetes. At the end of 8 wk, left ventricular (LV) hemodynamic assessment revealed compromised function in ND compared with the NN group. LV myocardial histology revealed increased collagen deposition in ND compared with the NN group, while electron microscopy showed a reduction in the viable mitochondrial fraction. Although the PKC-βII levels and activity were unchanged in the diabetic heart, the DAG levels were increased. With exercise training, the deterioration of LV structure and function in diabetes was attenuated. Notably, improved cardiac performance in training was associated with a decrease in myocardial DAG levels in diabetes. Exercise-induced benefits on cardiac performance in diabetes may be mediated by prevention of an increase in myocardial DAG levels.

Time-Dependent Alterations in Rat Macrovessels with Type 1 Diabetes

Vascular complications are associated with the progressive severity of diabetes, resulting in significant morbidity and mortality. This study quantifies functional vascular parameters and macrovascular structure in a rat model of type 1 diabetes. While there was no difference in the systemic arterial elastance (Ea) with 50 days of diabetes, changes were noted in the aorta and femoral artery including increased tunica media extracellular matrix content, decreased width of both the media and individual smooth muscle cell layers, and increased incidence of damaged mitochondria. Extracellular matrix proteins and elastin levels were significantly greater in the aorta of diabetic animals. These differences correlated with diminished matrix metalloprotease activity in the aorta of the diabetic animals. In conclusion, diabetes significantly altered the structure and ultrastructure of the aorta and femoral artery before systemic changes in arterial elastance could be detected.

Extracellular matrix motion and early morphogenesis.

For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale ‘total’ cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis. Summary: This Review considers the important, but often overlooked, role of the extracellular matrix in cell migration and tissue morphogenesis in amniotes.

Spatial anisotropies and temporal fluctuations in extracellular matrix network texture during early embryogenesis.

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks — (a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time-dependent rearrangements of a primordial biomaterial. We conclude that the ECM microenvironment changes markedly in time and space during the most important period of amniote morphogenesis—as determined by fluctuating textural properties.

Ribbon regulates morphogenesis of the Drosophila embryonic salivary gland through transcriptional activation and repression

Transcription factors affect spatiotemporal patterns of gene expression often regulating multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape/volume increases during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the changes in cell shape/volume in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib, we performed ChIP-seq analysis in embryos driving expression of GFP-tagged Rib specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites bound by Rib likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell growth and tissue shape in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that autoregulation of rib expression may be a key component of the SG morphogenetic gene network.

Identification of emergent motion compartments in the amniote embryo

Abstract The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ∼400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensens node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensens node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.