The functional role of cardiac non-neuronal cholinergic system in the diabetic heart

In type-2 diabetes mellitus (T2DM), insulin resistance and metabolic derangements reduce glucose transporter-4 (GLUT-4) expression to decrease glucose uptake, and hence the glucose oxidation in the diabetic heart. Further, the diabetic heart displays reduced basal myocardial energy status while diminished glucose oxidation further aggravates this condition in myocardial ischemia. Previous studies showed that normalization of glucose metabolism via increasing GLUT-4 expression attenuated diabetes-induced cardiac dysfunction. Cardiomyocytes possess a non-neuronal cholinergic system (NNCS) that consists of choline acetyltransferase (ChAT), choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AChE) to synthesize, release and degrade acetylcholine (ACh), respectively. The released ACh binds to type-2 muscarinic ACh receptor (M2AChR) and mediates pro-survival phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) /hypoxia-inducible factor1α (HIF1α) signaling cascade to promote glucose metabolism through increasing GLUT-4 expression in normoxic condition. However, the expression and function of cardiac NNCS are not known in the diabetic heart. Therefore, the main aim of this thesis was to examine the role of cardiac NNCS in the diabetic heart

Analysis of Subchondral Bone and Microvessels Using a Novel Vascular Perfusion Contrast Agent and Optimized Dual-Energy Computed Tomography

Osteoarthritis (OA), is a chronic debilitating disease that affects millions of individuals and is characterized by the degeneration of joint subchondral bone and cartilage. These tissue degenerations manifest as joint pain, limited range of joint motion, and overall diminished quality of life. Currently, the exact mechanism(s) and cause(s) by which OA initiates and progresses remain unknown. The multi-factorial complex nature of OA (i.e. age, diabetes, obesity, and prior injuries have all been shown to play a role in OA) contributes to the current lack of a cure or effective long-term treatment for OA. One re-emerging and interesting hypothesis revolves around the delicate homeostatic microvascular environment around the cartilage – an avascular tissue. The absence of blood vessels within cartilage stresses the importance of nutrient and oxygen delivery from the neighbouring synovium and subchondral bone. Currently, the effects of changes in the subchondral bone microvessel density on cartilage health remain unknown due to the difficulties in simultaneously studying dense bone and the associated small microvessels. Computed tomography (CT) is widely used in the diagnosis of OA, as the use of x-rays provide detailed images of the bone degeneration associated with OA. However, the study of microvessels using CT has been exceptionally difficult due to their small (\u3c 10 µm) size, lack of contrast from neighbouring soft tissues, and proximity to dense bone. The purpose of this thesis was to develop a novel dual-energy micro-computed tomography (DECT) compatible vascular perfusion contrast agent and the associated instrumentation to optimize DECT on pre-clinical, cone-beam micro-CT scanners. The combination of these two techniques would facilitate the simultaneous visualization and quantification of subchondral bone and microvessels within the bone underlining the cartilage (i.e. distal femoral epiphysis and proximal tibial epiphysis) of rats that have undergone an OA-induced surgery. Results gained from this study will further provide information into the role that microvessels may play in OA

Ischemic Stroke in Type II Diabetic Mice: Deregulation of SDF-1a/CXCR4 Axis

Type 2 diabetes mellitus is a major risk factor for ischemic stroke. Also diabetes is associated with poor outcome after stroke. Underlying mechanisms are however not fully understood. Alteration in the expression of the SDF-1a/CXCR4 axis, which is important for ischemic tissue repair, can be a probable cause. In this study, we have determined the expression of SDF-1a/CXCR4 in the brains of type II diabetic mice at basal and in response to ischemic stroke and have investigated a method for overexpression of SDF-1a in the brains of the diabetic mice. Adult male C57BLKS/J mice (db/db) of age 8 weeks were used as the murine model for type II diabetes and their age matched lean littermates served as controls (db/+). Microvascular density was first determined in the cerebral cortex of db/db diabetic mice by immunohistochemical analysis. Focal cerebral ischemia was induced by middle cerebral artery occlusion surgery (MCAO) in type 2 diabetic db/db mice and their controls. 48 hours after surgery, volume of ischemic damage was determined by TTC staining. The expression of SDF-1a and CXCR4 in the ischemic and non ischemic sides of brains of both the groups were determined using western blot and real time RT PCR. The db/db diabetic mice were injected with the vector, adeno associated virus 9 (AAV-SDF-1a) in the brain striatum and the overexpression of SDF-1a was determined by immunohistochemical analysis. Double immunohistochemistry was used to determine the localization of SDF-1a in brain after injection of the vector. The microvascular density in the cerebral cortex was reduced in db/db mice as compared with db/+ mice (p\u3c0.05). Volume of ischemic damage was significantly increased in db/db mice after focal cerebral ischemia (p\u3c 0.01). The levels of SDF-1a expression in both ischemic and non ischemic side of brain were reduced in db/db mice as compared with those in db/+ mice at mRNA (p\u3c 0.01) and protein level (p\u3c 0.01). The amount of CXCR4 expression was significantly reduced only in the ischemic side of the brains of db/db mice at protein level (p=0.001) and at m-RNA level (p=0.001). But in the non ischemic side, the expression of CXCR4 did not show any significant difference between the two groups. Immunohistochemical analysis showed overexpression of SDF-1a in the striatum receiving the microinjection of AAV-SDF-1a and double immunohistochemistry showed SDF-1a to be localized in the glial cells of the cerebral striatum after microinjection. The results indicate that microvascular density is reduced and ischemia induced cerebral damage is enlarged in diabetes which may be linked to the impaired expression of hypoxia regulated SDF-1a/CXCR4 axis after ischemic stroke in diabetes and vector mediated over expression of SDF-1a in the brain can be a novel therapeutic technique for treating ischemic stroke in diabetics

Molecular Mechanism of Congenital Heart Disease and Pulmonary Hypertension

This open access book focuses on the molecular mechanism of congenital heart disease and pulmonary hypertension, offering new insights into the development of pulmonary circulation and the ductus arteriosus. It describes in detail the molecular mechanisms involved in the development and morphogenesis of the heart, lungs and ductus arteriosus, covering a range of topics such as gene functions, growth factors, transcription factors and cellular interactions, as well as stem cell engineering technologies. The book also presents recent advances in our understanding of the molecular mechanism of lung development, pulmonary hypertension and molecular regulation of the ductus arteriosus. As such, it is an ideal resource for physicians, scientists and investigators interested in the latest findings on the origins of congenital heart disease and potential future therapies involving pulmonary circulation/hypertension and the ductus arteriosus

Major Collateral Vessels Develop from Pre-existing Small Arteries through RAC2/NOX2 Independent Mechanisms

Indiana University-Purdue University Indianapolis (IUPUI)There is no consensus on which vascular segment or what size of vessels is most important in the process of collateral growth, the degree to which these vessels can enlarge, or the mechanisms that mediate collateral vessel expansion and its impairment. Chapter I identifies the major collateral vessels that develop in response to femoral arterial occlusion in the pig, rat, and mouse hindlimbs for comparison to humans. Pre-existent small named arteries enlarged ~2-3-fold to become the major collateral vessels in each species, these major collaterals displayed characteristics similar to large arteries experiencing flow-mediated outward remodeling, and important differences in vascular wall thickness were observed between rodents and pigs. Chapter II utilized Rac2-/- and Nox2-/- mice to investigate the hypothesis that Nox2-NAD(P)H oxidase is required for major collateral growth subsequent to femoral arterial occlusion. Previous studies suggest bone marrow cell (BMC)-derived reactive oxygen species (ROS) produced by the Nox2 subunit of NAD(P)H oxidase plays an important role in neovascularization and recovery of hindlimb perfusion subsequent to femoral arterial occlusion; but did not investigate collateral growth. The hematopoietic cell restricted protein Rac2 has been shown to bind to and activate Nox2-NAD(P)H oxidase and Rac2-/- and Nox2-/- leukocytes display impaired ROS related functions. The data demonstrated that Rac2 and Nox2 are not essential for major collateral growth, but both are important for the recovery of hindlimb perfusion and preservation of distal tissue morphology. Chapter III investigated BMC and antioxidant therapy in the age-related impairment of collateral growth. Aging, like all cardiovascular disease risk factors is associated with elevated ROS and impaired collateral growth. Studies also suggest BMCs promote collateral growth by secreting paracrine factors but elevated ROS may affect the efficacy of BMCs. The data revealed that neither BMC injection nor antioxidant therapy via apocynin enhanced the process of major collateral artery growth in aged mice

Distinctive Alterations in Microvascular Function Due to Multiple Common Morbidities

In this thesis, we investigated the role of multiple common cardiovascular risk factors on the development of microvascular dysfunction and left ventricular diastolic dysfunctio

Untersuchung der Wirkung des Moringa oleifera-Extraktes auf den Tonus der glatten Muskulatur

In der vorliegenden Studie wurde die Wirkung des Moringa oleifera-Extraktes auf die glatte Muskulatur verschiedenen Organursprungs getestet. Am Magen entfaltet Moringa eine exzitatorische Reaktion. Die Messung der Wirkung auf die glatte Muskulatur der Herzkranzgefäße ergaben biphasische, aber auch konstriktorische Reaktionen. In den Experimenten an der thorakalen Rattenaorta konnte gezeigt werden, dass Moringa als vorrangige Wirkung eine Vasodilatation hervorruft. Diese ist von der Funktionsfähigkeit des Endothels abhängig und wird über die Produktion und den Signalweg des NOs vermittelt

Micro-CT based characterisation of changes to the vascular network following closed soft tissue trauma and cryotherapy

This project has investigated how the architecture of the blood vessels supplying nutrients to skeletal muscles is affected by muscle contusion injuries, and how it changes during healing with or without initial treatment of the injury by icing. In order to do this, we used contrast agents to visualise blood vessels in 3D with micro-computed tomography imaging. This research significantly contributes to the fields of orthopaedics, traumatology and sports medicine, as it improves our understanding of muscle contusion injuries. Furthermore, the methods developed in this thesis may help to improve the diagnosis and monitoring of these injuries