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Osteogenic Regulatory Mechanisms Activated By Pressure In Aortic Heart ValveGamez, Carol Andrea Pregonero 11 December 2009 (has links)
Calcific aortic valve disease (CAVD) is the most common cause of aortic valve failure and replacement in the elderly population, affecting 25% of the population over 65 years of age. Current pharmacological approaches for preventing the onset and progression of calcific aortic valve disease have not shown consistent benefits in clinical studies. Differentiation of valvular interstitial cells (VICs) into osteoblast–like cells is an integral step in the calcification process. Although clinical evidence suggests hypertension as a potential candidate contributing to the development of CAVD, the underlying molecular mechanisms that cause de-differentiation remain unclear. The present study investigates the role of elevated cyclic pressure in modulating osteoblast differentiation pathways in VICs in vitro. We used a combination of systems biology modeling and pathway-based analyses to identify novel genes and molecular mechanisms that are activated in valve tissue during exposure to elevated pressure conditions. Our results show that elevated pressure induces a gene expression pattern in valve tissue that is considerably similar to that seen in CAVD, underlining the key role of hypertension as an initiating factor in the onset of pathogenesis. In addition, our analysis revealed a set of genes that was not previously known to be regulated in valve tissue in a pressure dependent manner. Currently, the molecular mechanisms involved in CAVD and their associations with changes in local mechanical environment are poorly understood, and thus a better understanding of the cell based process mediating CAVD progression will improve our ability to develop potential medical therapies for this disease.
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ON THE ORIGIN AND FUNCTION OF WAXING AND WANING IN PACEMAKER ACTIVITY IN THE SMALL INTESTINEPawelka, Andrew J. 15 January 2015 (has links)
<p>The small intestine of the gastrointestinal tract displays a variety of motor patterns involved in the mixing, digestion, and propulsion of luminal content. Ultimately, it is the co-ordinated effort of smooth muscle contraction influenced by neural and myogenic stimulation that facilitate these motor patterns. While neural input from the enteric nervous system (ENS) and slow wave producing activity of the peacemaking interstitial cells of Cajal (ICC) in the myenteric plexus (ICC-MP) are key players in the manipulation of smooth muscle cells, the mechanisms behind the onset the segmentation motor pattern are currently unknown. I have demonstrated with intracellular recordings of electrical activity from circular smooth muscle cells, the same nutrient stimulants used to induce the segmentation motor pattern in whole organ preparations evokes the waxing waning phenomenon of the smooth muscle slow wave. Through the use of continuous wavelet transformation analysis on nutrient induced waxing waning, it was determined that the induction of a rhythmic low frequency component is responsible for the generation of waxing waning. Stimulated low frequency activity after methylene blue mediated elimination of ICC-MP slow wave activity suggested the low frequency component did not originate from the ICC-MP. The hypothesis emerged that the ICC of the deep muscular plexus (ICC-DMP), on the opposite side of the circular muscle thickness to the ICC-MP, were responsible for the low frequency oscillations. ICC-DMP networks in close physical proximity to nerve fibers were found to be under tonic inhibited by nitric oxide, and to respond to substance P stimulation. Both alleviation of the inhibition and stimulus by tachykinergic neurotransmission induced the low frequency component and waxing waning. The ENS and myogenic pacemakers play an important role in stimulating the segmentation motor pattern. ICC-DMP are the pacemakers responsible for generation of the low frequency component involved in waxing waning and segmentation.</p> / Master of Science (MSc)
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Electrophysiology of interstitial cells of CajalWright, George January 2017 (has links)
This thesis focuses on elucidating the electrical mechanisms underlying excitation of small intestinal and colonic smooth muscle initiated by interstitial cells of Cajal (ICC). All the ICC subtypes are involved in the orchestration, generation, and/or transmission of electrical signals to smooth muscle to pace gut motor patterns. Some ICC types have intrinsic activity leading to omnipresent rhythmic changes in smooth muscle excitability; others respond to stimuli, inducing pacemaker activity as required. Together they orchestrate motor patterns such as propulsion and segmentation, essential functions of the gut. To study ICC electrophysiology, I utilized patch clamping to record ion channel currents from single intestinal ICC and sharp microelectrodes to record colonic smooth muscle membrane potentials. I have made several discoveries contributing to our understanding of ICC electrophysiology. Firstly, my research increased our understanding of the properties of intrinsic pace-maker activity. I showed that maxi Cl– channels from small intestinal ICC make a significant contribution to slow wave depolarization triggered by intracellular calcium. Secondly, I showed that colonic intramuscular ICC (ICC-IM) selectively express KV7.5 channels, which are suppressed by cholinergic agonists, meaning that excitatory stimuli triggering acetylcholine release deactivate KV7.5 channels, leading to increased excitability. Thirdly, I have shown that the bile acid chenodeoxycholic acid and the nitric oxide donor sodium ni-troprusside both induce pacemaker activity, rhythmic transient depolarisations in mouse colonic muscle, which led to the hypothesis that nitrergic nerves are involved in generating inducible myenteric plexus ICC (ICC-MP) pacemaker activity. It is only when ICC are suitably stimulated by intracellular processes such as rhythmic Ca2+ transients or extracellular signalling from neurotransmitters or small molecules, that ICC produce membrane potential rhythmicity, required for generation of intrinsic slow waves, low-frequency rhythmic transient depolarisations and transmission of excitation into the muscle. / Thesis / Doctor of Philosophy (PhD) / The gut is essential for digestion and absorption of food. The gut has special cells called interstitial cells of Cajal (ICC), which control the contractions of the gut muscle. ICC are pacemaker cells, like those that pace heart beats. To pace gut muscle contractions, ICC generate electrical signals which cause the muscle to contract in an organized rhythmic manner, which promotes mixing or propulsion of gut contents, called motility. I used tiny electrodes to record electrical activity from ICC or gut muscle, to improve our understanding of how ICC pacemaker activity controls motility. My research characterised ion channels, which are microscopic protein pores that allow cells to make electrical currents, that enable generation of pacemaker signals by ICC. I also investigated activation of ICC electrical activity that causes propulsive colonic motility. This will hopefully lead to treatment improvements for patients with motility disorders in the future.
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Tissue-engineered canine mitral valve constructs as in vitro research models for myxomatous mitral valve diseaseLiu, Mengmeng January 2014 (has links)
Myxomatous mitral valve disease (MMVD) is one of the most common degenerative cardiac diseases affecting humans and dogs; however, its pathogenesis is not completely understood. This study focussed on developing tissue-engineered fibrin based canine mitral valve constructs, which can be used as an in vitro platform to study the pathogenesis of MMVD. Prior to three dimensional (3D) construct fabrication, primary canine mitral valve endothelial cells (VECs) and valve interstitial cells (VICs) were isolated, cultured and characterized utilising a variety of techniques. Moreover, preliminary experiments were carried out to optimise the purity of VEC cultures. It is uncertain if canine MMVD is initiated by long term shear stress damage to the valve endothelium or from abnormalities of VICs. To investigate both hypotheses, three types of models were produced using fibrin/based 3D culture techniques: healthy VEC-VIC co-culture (Type 1); healthy VEC-diseased VIC co-culture (Type 2); healthy VEC-VIC co-culture with endothelial damage during culture (Type 3). Histological examination demonstrated partial native tissue-like morphology of the 3D constructs. Results suggest that current static cultured constructs express MMVD markers irrespective of using healthy or diseased VICs. Simple mechanical stimulation was found to regulate VIC activity in the 3D models. Endothelial damage resulting in VIC phenotypic activation (a change typically observed in MMVD), and decreased mechanical tension appeared to be a negative regulator of this effect. Moreover, there appears to be heterogeneity in the activated VIC population. Additionally, distinct advanced glycation end product (AGE) carboxymethyllysine (CML) expression was found in canine MMVD valves, which suggesting this biochemical compound (known to affect long living protein) might be a putative regulator of MMVD pathogenesis. The role of CML in MMVD can be further investigated utilizing current 3D static mitral valve construct model in future studies. Lastly a prototype dynamic tubular construct and a customised bioreactor system were developed. Preliminary data suggest the feasibility of tubular construct fabrication and endothelialisation, which provides foundation for future dynamic conditioning experiments and will allow examination of the role of endothelial shear stress in triggering MMVD. In summary, this project successfully developed fibrin based canine mitral valve constructs. It is believed they are promising models for MMVD research, allowing new insights in understanding MMVD pathogenesis.
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Pathology of Calcific Aortic Valve Disease: The Role of Mechanical and Biochemical Stimuli in Modulating the Phenotype of and Calcification by Valvular Interstitial CellsYip, Cindy Ying Yin 16 March 2011 (has links)
Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that are mediated by valvular interstitial cells (VICs). VICs undergo pathological differentiation during the progression of valve calcification; however the factors that regulate cellular differentiation are not well defined. Most commonly recognized are biochemical factors that induce pathological differentiation, but little is known regarding the biochemical factors that may suppress this process. Further, the contribution of matrix mechanics in valve pathology has been overlooked, despite increasing evidence of close relationships between changes in tissue mechanics, disease progression and the regulation of cellular response. In this thesis, the effect of matrix stiffness on the differentiation of and calcification by VICs in response to pro-calcific and anti-calcific biochemical factors was investigated. Matrix stiffness modulated the response of VICs to pro-calcific factors, leading to two distinct calcification processes. VICs cultured on the more compliant matrices underwent calcification via osteoblast differentiation, whereas those cultured on the stiffer matrices were prone to myofibroblast differentiation. The transition of fibroblastic VICs to myofibroblasts increased cellular contractility, which led to contraction-mediated, apoptosis-dependent calcification. In addition, C-type natriuretic peptide (CNP), a putative protective molecule against CAVD, was identified. CNP supressed myofibroblast and osteoblast differentiation of VICs, and thereby inhibited calcification in vitro. Matrix stiffness modulated the expression of CNP-regulated transcripts, with only a small number of CNP-regulated transcripts not being sensitive to matrix mechanics. These data demonstrate the combined effects of mechanical and biochemical cues in defining VIC phenotype and responses, with implications for the interpretation of in vitro models of VIC calcification and possibly disease devleopment. The findings from this thesis emphasize the necessity to consider both biochemical and mechanical factors in order to improve fundamental understanding of VIC biology.
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An investigation into gastric myoelectrical activity in response to drug treatment during ageing and in a mouse model of Alzheimer's disease. / CUHK electronic theses & dissertations collectionJanuary 2013 (has links)
Wang, Hui Chuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 184-202). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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Diuretic, natriuretic, and vasodepressor activity of a lipid fraction enhanced in medium of cultured mouse medullary interstitial cells by a selective FAAH inhibitorDaneva, Zdravka P 01 January 2019 (has links)
The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not well understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF-3845. Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm-absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically-instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 AU/10 min) stimulated diuresis and natriuresis, whereas at higher doses, these parameters returned toward baseline but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, GFR and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urinary parameters. Genetic or pharmacological ablation of the cannabinoid type 1 receptors in mice completely abolished the diuretic and vasodepressor properties of intramedullary infused PIP, suggesting that the PF-3845-induced product requires the presence of CB1 receptors in order to elicit its renal effects. In a radioactive competition binding assay, using Chinese hamster ovary cells expressing CB1 receptors, PIP successfully displaced the CB1 selective inverse agonist [3H] SR141716A, revealing that the lipid extract was able to compete for binding to CB1 receptors. Finally, we investigated the tubular location of diuretic activity that the PF-3845-induced lipid fraction exhibits. In a renal function in vivo experiment, we pre-treated anesthetized mice with an intramedullary infusion of one of four well-known diuretics. This procedure was followed by an intramedullary infusion of PIP (1AU). Only inhibition of the proximal tubule sodium reabsorption diminished the diuretic activity of the PF-3845-induced product, suggesting that the lipid fraction requires a physiologically intact proximal tubular reabsorption mechanism for it to produce diuresis. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.
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Hyaluronan and Renal Fluid Handling : Studies during Normal and Pathological Conditions of Renal FunctionGöransson, Viktoria January 2001 (has links)
<p>The kidney is the major organ responsible for the regulation of the composition and volume of the body fluids, which is essential for homeostasis. The glycosaminoglycan hyaluronan (HA), with extreme water-binding capacity, is present in the interstitium of the kidney with a heterogenous distribution. The importance of HA in renal water-handling is unknown and was the focus of the present investigation.</p><p>Acute water-loading in rats caused the amount of papillary HA to increase and during water deprivation, the amount was reduced. Gerbils, with extreme urine concentrating capacity, have less HA in the renal papilla in normal conditions and responded diametrically different to water-loading (reduction in HA). Renomedullary interstitial cells (RMICs), which are probably the main producers of HA in the renal medulla, were cultured at different media osmolalities to mimic the milieu of the medulla during variations in the water balance. The amount of HA found in the media was decreased at high osmolalities and increased at low osmolalities, thereby strengthening the <i>in vivo</i> results. CD44, an HA-receptor involved in the uptake and degradation of HA, was expressed on RMICs in an osmolality dependent manner. During high media osmolality, the CD44 expression increased and at lower osmolalities, the opposite occurred, probably due to the need for uptake and degradation of HA.</p><p>Renal ischemia-reperfusion injury causes a cortical accumulation of HA, up-regulation of CD44, and a depression of functional parameters. The time periods of ischemia correlated with the accumulation of HA which, in turn, was inversely correlated to GFR. Hyaluronidase injections in this setting failed to reduce HA levels and significantly improve renal function.</p><p>In conclusion, the results from the present study suggest an important role for HA and RMICs in renal water-handling and that the intrarenal distribution of HA is altered after ischemia-reperfusion injury, which correlates with renal dysfunction.</p>
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Hyaluronan and Renal Fluid Handling : Studies during Normal and Pathological Conditions of Renal FunctionGöransson, Viktoria January 2001 (has links)
The kidney is the major organ responsible for the regulation of the composition and volume of the body fluids, which is essential for homeostasis. The glycosaminoglycan hyaluronan (HA), with extreme water-binding capacity, is present in the interstitium of the kidney with a heterogenous distribution. The importance of HA in renal water-handling is unknown and was the focus of the present investigation. Acute water-loading in rats caused the amount of papillary HA to increase and during water deprivation, the amount was reduced. Gerbils, with extreme urine concentrating capacity, have less HA in the renal papilla in normal conditions and responded diametrically different to water-loading (reduction in HA). Renomedullary interstitial cells (RMICs), which are probably the main producers of HA in the renal medulla, were cultured at different media osmolalities to mimic the milieu of the medulla during variations in the water balance. The amount of HA found in the media was decreased at high osmolalities and increased at low osmolalities, thereby strengthening the in vivo results. CD44, an HA-receptor involved in the uptake and degradation of HA, was expressed on RMICs in an osmolality dependent manner. During high media osmolality, the CD44 expression increased and at lower osmolalities, the opposite occurred, probably due to the need for uptake and degradation of HA. Renal ischemia-reperfusion injury causes a cortical accumulation of HA, up-regulation of CD44, and a depression of functional parameters. The time periods of ischemia correlated with the accumulation of HA which, in turn, was inversely correlated to GFR. Hyaluronidase injections in this setting failed to reduce HA levels and significantly improve renal function. In conclusion, the results from the present study suggest an important role for HA and RMICs in renal water-handling and that the intrarenal distribution of HA is altered after ischemia-reperfusion injury, which correlates with renal dysfunction.
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Pathology of Calcific Aortic Valve Disease: The Role of Mechanical and Biochemical Stimuli in Modulating the Phenotype of and Calcification by Valvular Interstitial CellsYip, Cindy Ying Yin 16 March 2011 (has links)
Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that are mediated by valvular interstitial cells (VICs). VICs undergo pathological differentiation during the progression of valve calcification; however the factors that regulate cellular differentiation are not well defined. Most commonly recognized are biochemical factors that induce pathological differentiation, but little is known regarding the biochemical factors that may suppress this process. Further, the contribution of matrix mechanics in valve pathology has been overlooked, despite increasing evidence of close relationships between changes in tissue mechanics, disease progression and the regulation of cellular response. In this thesis, the effect of matrix stiffness on the differentiation of and calcification by VICs in response to pro-calcific and anti-calcific biochemical factors was investigated. Matrix stiffness modulated the response of VICs to pro-calcific factors, leading to two distinct calcification processes. VICs cultured on the more compliant matrices underwent calcification via osteoblast differentiation, whereas those cultured on the stiffer matrices were prone to myofibroblast differentiation. The transition of fibroblastic VICs to myofibroblasts increased cellular contractility, which led to contraction-mediated, apoptosis-dependent calcification. In addition, C-type natriuretic peptide (CNP), a putative protective molecule against CAVD, was identified. CNP supressed myofibroblast and osteoblast differentiation of VICs, and thereby inhibited calcification in vitro. Matrix stiffness modulated the expression of CNP-regulated transcripts, with only a small number of CNP-regulated transcripts not being sensitive to matrix mechanics. These data demonstrate the combined effects of mechanical and biochemical cues in defining VIC phenotype and responses, with implications for the interpretation of in vitro models of VIC calcification and possibly disease devleopment. The findings from this thesis emphasize the necessity to consider both biochemical and mechanical factors in order to improve fundamental understanding of VIC biology.
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