Doctor of Philosophy / Department of Anatomy and Physiology / Timothy I. Musch / Nitric oxide (NO) is synthesized via distinct NO synthase (NOS) enzymes and constitutes an essential cardiovascular signaling molecule. Whereas important vasomotor contributions of endothelial NOS (eNOS) have been well-described, the specific vasomotor contributions of nNOS-derived NO in healthy subjects during exercise are unknown. The purpose of this dissertation is to test the global hypothesis that nNOS-derived NO is a critical regulator of exercising skeletal muscle vascular control. Specifically, we utilized the selective nNOS inhibitor S-methyl-L-thiocitrulline (SMTC) to investigate the effects of nNOS-derived NO on skeletal muscle vascular function within established rodent models of exercise performance. The first investigation (Chapter 2) identifies that nNOS inhibition with SMTC increases mean arterial pressure (MAP) and reduces rat hindlimb skeletal muscle blood flow at rest whereas there are no effects during low-speed (20 m/min) treadmill running. In Chapter 3 it is reported that nNOS inhibition with SMTC reduces blood flow during high-speed treadmill running (>50 m/min) with the greatest relative effects found in highly glycolytic fast-twitch muscles and muscle parts. Chapter 4 demonstrates that nNOS-derived NO modulates contracting skeletal muscle blood flow (increases), O2 consumption (VO2, increases), and force production (decreases) in the rat spinotrapezius muscle and thus impacts the microvascular O2 delivery-VO2 ratio (which sets the microvascular partial pressure of O2, PO2mv, and represents the pressure head that drives capillary-myocyte O2 diffusion). In Chapter 5 we report that systemic administration of the selective nNOS inhibitor SMTC does not impact lumbar sympathetic nerve discharge. This reveals that the SMTC-induced peripheral vascular effects described herein reflect peripheral nNOS-derived NO signaling as opposed to centrally-derived regulation. In conclusion, nNOS-derived NO exerts exercise-intensity and muscle fiber-type selective peripheral vascular effects during whole-body locomotor exercise. In addition, nNOS-derived NO modulates skeletal muscle contractile and metabolic function and, therefore, impacts the skeletal muscle PO2mv. These data identify novel integrated roles for nNOS-derived NO within healthy skeletal muscle and have important implications for populations associated with reduced NO bioavailability and/or impaired nNOS structure and/or function specifically (e.g., muscular dystrophy, chronic heart failure, advanced age, etc.).

M.Tech. (Chiropractic) / Please refer to full text to view abstract

Increasing numbers of old and sick patients who are no longer eligible for prolonged invasive implantation surgery have encouraged many researchers to investigate the development of a Mechanical Circulatory Support (MCS) device with more reliability and less possible invasive complications, which would benefit the majority of patients. This thesis will test experimentally and numerically the feasibility of installing an MCS device, as a bridge to destination, in the descending aorta, in a series configuration with the heart. To this end, a multi-chamber Simulator of the Cardio-Vascular blood-flow Loop (SCVL) was designed to simulate the in-vitro flow rates, pressures and other parameters representing normal and diseased conditions of the human cardiovascular system. The multi-chamber SCVL includes models for all four chambers of the heart, and the systemic as well as the pulmonic circulations. Next, a comprehensive study was conducted using the SCVL system to compare the novel in-series placement of the pump, in the descending aorta, with traditional in-parallel placements. Then, a comprehensive numerical study was conducted using the modified Concentrated Lumped Parameter (CLP) model developed by the same team. The numerical results are compared and verified by the experimental results under various conditions. The results for the pump installed in the descending aorta show that the pressure drop, upstream of the pump, facilitates the cardiac output as a result of after-load reduction. However, at the same time the generated pressure drop at the proximal part of the descending aorta induces a slight drop in the carotid perfusion which will be autoregulated by the brain in a native system. Further, the pressure rise downstream of the pump improves the blood perfusion in the renal artery. The pulse wave analysis show that the placement of the pump in the descending aorta leads to improved pulsatility which is beneficial for end-organ functionality in the native cardiovascular system.

Intracranial pressure (ICP) is an important monitoring modality in the clinical management of several neurological diseases carrying the risk of fatal intracranial hypertension. However, this parameter is not always considered due to its invasive assessment. In this scenario, a non-invasive estimation of ICP (nICP) may be essential, and indeed it has become a Holy Grail in Clinical Neurosciences: extensively searched, albeit never found. This thesis is devoted to the assessment, applications and development of transcranial Doppler (TCD)-based non-invasive methods for ICP and cerebral perfusion pressure (CPP) monitoring. The thesis is divided into three sections: I) The accuracy of existing TCD-based nICP estimators in various scenarios of varying ICP (traumatic brain injury, rise of ICP during plateau waves, and rise in ICP induced by infusion of cerebrospinal fluid during infusion test). The estimators of nICP consisted of a mathematical black box model, methods based on non-invasive CPP, and a method based on TCD pulsatility index. II) The feasibility of the best performing nICP estimator in clinical practice, including patients with closed TBI and brain midline shift, patients with acute liver failure during liver transplant surgery, and patients during non-neurosurgical surgery in the beach chair position. III) The description and assessment of a novel methodology for non-invasive assessment of cerebral perfusion pressure (nCPP) based on spectral arterial blood volume accounting. As main results, TCD-based non-invasive methods could replicate changes in direct ICP across time confidently, and could provide reasonable accuracy in comparison to the standard invasive techniques. Furthermore, in feasibility studies, nICP in association with other TCD physiological parameters provided a comprehensive interpretation of cerebral haemodynamics in conditions presenting impairment of cerebral blood flow circulation. The new method of nCPP estimation could identify changes in CPP across time reliably in conditions of decreasing and increasing CPP. These findings support the use of TCD-based nICP methods in a variety of clinical conditions requiring management of ICP and brain perfusion. More importantly, the low costs associated with nICP methods, since TCD is a widely available medical device, could contribute to its widespread use as a reliable alternative for ICP monitoring in everyday clinical practice.

Ho Sin Yee, Stella. / "August 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 167-193). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Emphysema, a subset of COPD, occurs due to an abnormal inflammatory response to noxious gases or particles leading an influx of immunologic cells. Recent studies have demonstrated endothelial dysfunction in COPD subjects and are suggestive of a vascular phenotype present in COPD that is not fully characterized. We hypothesize that processes affecting the pulmonary vasculature lead to early changes important in the pathogenesis of COPD. This work focuses on the use of multidetector-row computed tomography (MDCT)-based measures of pulmonary blood flow (PBF), mean transit time (MTT) and pulmonary vascular volume (TPVV) to gain new insights into vasculature-related changes present in COPD. As a precursor to using perfusion MDCT imaging to phenotype lung disease, we demonstrated good regional correlation of PBF measurements obtained with MDCT imaging and fluorescent microspheres (FMS) at a FMS piece size resolution of 1.9 cm3 and regional volume level of 8-10 cm3. Additionally, we developed an ex vivo perfusion system, and applied quantitative image analysis techniques to study the lung preparation's stability over 120 minutes. We further validated CT-based PBF and MTT measurements by demonstrating physiologically appropriate responses to a range of flow rates with this new system. Finally, quantitative MDCT-based measurements were used to characterize a novel phenotype of emphysema and test hypotheses regarding vasculature-related changes in smokers and COPD subjects. We demonstrated increased heterogeneity in regional MTT and PBF measurements in smokers with preclinical emphysema compared with smokers with normal lung function and imaging studies and nonsmokers. This data is supportive of the notion that inflammatory-based vascular responses to hypoxia are occurring in smokers susceptible to COPD, but are successfully blocked in smokers without signs of emphysema. A new CT-based measure, TPVV, was studied and we demonstrate its association with total lung volume and body size metrics. TPVV measurements correlated with measures of COPD severity. A trend linking increased TPVV with increased endothelial dysfunction was observed, suggesting that pathological changes of COPD have an effect on the pulmonary vasculature. This work demonstrates the importance of functional information that can compliment structural, anatomical information to answer questions based on the lung physiology and pathological disease processes.

Abstract not available

<p>Positron Emission Tomography, PET, is a noninvasive medical imaging technique to get functional information of the kinetics of radioactive compound injected in the body. The data used in this thesis comes from a total of five H₂¹⁵O PET studies of one patient. This was done in order to study the blood flow in liver metastasis of the patient, before and after treatment.</p><p>A one compartment model was used to do the ROI based analyses. With a least square method in Matlab the unknown parameters in the model, such as the kinetic rate constants, the dispersion and the fraction of blood in the tissue, was extracted. Also a brief analysis of different parts of the metastases, edge and center, was done to see the variations within the metastases. The results show some increase of the blood flow after the treatment, and two of the three studied metastases showed a distinct difference of the activity in the center versus the edge. Given in the thesis are also some basic PET and compartmental modeling theory.</p> / <p>Positronemissionstomografi, PET, är en icke-invasiv medicinsk bildteknik för att få funktionell information om kinetiken av radioaktiva föreningar injicerade i kroppen. Det data som används i denna kandidatuppsats kommer från totalt fem H₂¹⁵O PET-studier av en patient. Detta gjordes för att studera blodflödet i levermetastaser hos patienten före och efter behandling.</p><p>En 1-kompartmentmodell användes för att göra ROI-baserade analyser. Med en minsta kvadrat-metod i Matlab kunde de okända parametrarna i modellen, såsom den kinetiska hastighetskonstanten, spridningen och andelen blod i vävnaden, fås ut. En kort analys gjordes också av olika delar av metastaserna, kanten och mitten, för att se variationerna inuti metastaserna. Resultaten visar en viss ökning av blodflödet efter behandlingen, och två av de tre studerade metastaser visade en tydlig skillnad av aktiviteten i mitten jämfört med kanten. I rapporten ges också grundläggande teori om bland annat PET och kompartmentmodellering.</p>

Tumors need the development of new vessels from the pre-existing vasculature to bring nutrients and oxygen to the whole tumor mass. The tumor vascular network is known to be poorly functional due to architectural and functional abnormalities. The end result is an inadequate and heterogeneous tumor perfusion leading to the development of tumor hypoxia. From a therapeutic perspective, hypoxia is a source of radioresistance and the dysfunctional perfusion hampers drug delivery. Historically, tumor hypoxia refers to chronic hypoxia (or diffusion-limited hypoxia) that results from the increasing distance between O2-consuming cells and blood vessels due to the high metabolic rate of tumor cells. Many studies have demonstrated the impact of chronic hypoxia on the clonal selection of tumor cells resistant to conventional anti-cancer therapies. Growing evidence for the existence of another form of hypoxia caused by heterogeneities in tumor perfusion, namely acute or perfusion-limited hypoxia, plead however for a non-genetic source of phenotype conversion reaching not only tumor cells but also the tumor vasculature and in particular endothelial cells. In the cardiovascular field, the cyclic exposure to different pO2 levels is known to precondition cardiac myocytes to resist more prolonged ischemic insults. We hypothesized that this concept of myocardium preconditionning to promote the resistance vs pro-apoptotic stresses could be translated in tumors. Indeed, intermittent hypoxia in tumors is nothing else than cyclic changes in pO2 and radio- and chemotherapy can be viewed as pro-apoptotic stresses that the tumor can face. In particular, in the case of the tumor vasculature, the resistance could be a capacity to re-initiate angiogenesis after treatment. Radioresistance would be further potentiated since low pO2 is per se associated to reversibility of the damages. Also, since intermittent hypoxia is thought to be due in part to fluctuations in tumor blood flow (TBF), access of chemotherapy to the tumor could also further participate to chemoresistance. To address the above hypotheses, we first aimed to explore the extent and the origin of TBF fluctuations in tumor mouse models and to determine whether therapeutic modulation of such potential TBF heterogeneities could improve the efficacy of chemotherapy. We then more directly examined whether and how intermittent hypoxia could influence endothelial cell survival and modulate resistance to radiotherapy. We also took advantage of this study to dissect the molecular mechanisms driving the phenotype conversion of endothelial cells exposed to intermittent hypoxia. Finally, because VEGF plays a major role in hypoxia-mediated angiogenesis but also regulates major pro-survival pathways in endothelial cells, we evaluated the potential role of caveolin as a new therapeutic target to tackle EC resistance. Caveolin is, indeed, a key structural protein recently documented to interact with many downstream targets of VEGF. 1. To explore the extent and the origin of TBF fluctuations in tumor mouse models and to determine whether therapeutic modulation of such potential TBF heterogeneities could improve chemotherapy. We focused this part of the work on the vascular tone modulator endothelin-1. Indeed, this peptide is over-expressed in many mouse and human tumors where it is documented to act as a mitogenic factor in both para- and autocrine manners. Endothelin-1 is also a potent vasoconstrictor acting through the ETA receptors located on VSMCs. In our lab, we previously showed that over-expression of endothelin-1 in tumors accounted for the development of a myogenic tone within the tumor vasculature. We have now documented that an ETA receptor antagonist induces the relaxation of microdissected tumor arterioles and selectively and quantitatively increases tumor blood flow in experimental tumor models. We also combined dye staining of functional vessels, fluorescent microsphere-based mapping, and magnetic resonance imaging to identify heterogeneities in tumor blood flow and to examine the reversibility of such phenomena. We showed that administration of an ETA receptor antagonist reduces the extent of underperfused tumor areas, proving the key role of vessel tone variations in tumor blood flow heterogeneity. We also provided evidence that ETA antagonist could improve the access of cyclophosphamide to the tumor compartment and thereby induces a significant tumor growth delay. 2. To examine whether and how intermittent hypoxia could influence endothelial cell survival and modulate resistance to radiotherapy. To dissect the mechanisms driving the phenotype conversion of endothelial cells exposed to intermittent hypoxia. This second part of our work, is a comprehensive investigation of the consequences of intermittent hypoxia, as caused by TBF heterogeneities, on the endothelial cell phenotype. First, we postulated that intermittent hypoxia (IH) favors endothelial cell (EC) survival, thereby extending the concept of hypoxia-driven resistance to the tumor vasculature. We showed that exposing EC to cycles of hypoxia/re-oxygenation reduces radiation-induced cell death and promotes angiogenesis. In contrast, prolonged hypoxia failed to achieve such protection and even appeared deleterious. We also observed that although HIF-1£ is completely degraded during each re-oxygenation, its abundance is paradoxically found higher at each new hypoxic challenge. Moreover, the use of siRNA targeting HIF-1£ pointed out that HIF-1ƓÑ accumulation account for the increased resistance of EC to radiotherapy. Finally, we extended this concept in vivo by forcing IH in tumor-bearing mice and found that it is associated with less radiation-induced apoptosis within both the vascular and the tumor cell compartments (vs normoxia or prolonged hypoxia). Next, we focused our work on the underlying mechanisms of EC phenotype conversion exposed to IH and particularly on potential actors that may favor HIF-1£ accumulation during IH. Prolylhydroxylases (PHD), MAPK and PI3K/Akt pathways as well as eNOS are known to regulate HIF-1£ abundance and transcriptional activity. We documented that PHD2 and PHD3 abundance are slightly decreased during IH, whereas prolonged hypoxia increases PHD3 expression in EC. We then showed that, ERK, Akt as well as eNOS were phosphorylated during reoxygenation periods of the IH protocol. We also used specific inhibitors of these cascades (i.e. PD98059, LY294002 and L-NAME, respectively), to evaluate their specific impact on HIF-1£ abundance and performed clonogenic assays to evaluate their consequences on EC survival. We showed that although, PD98059 and LY294002 sensitizes EC to pro-apoptotic stresses, only the PI3K/Akt inhibitor abrogates the HIF-1£ signal during IH. Conversely, L-NAME, a non-specific NOS-inhibitor, appears to potentiate the expression of HIF-1£ and to favor the EC survival. 3. To identify new therapeutic targets to prevent endothelial cell resistance by studying VEGF signaling, the major pro-survival and pro-angiogenic growth factor in endothelial cells. Because VEGF plays a central role in hypoxia-mediated angiogenesis and cell survival, the VEGF signaling cascade is a an obvious therapeutic target. To more specifically identify the pathways leading to cell survival and the resistance phenomena that we observed in response to intermittent hypoxia, a careful dissection of the downstream VEGF signaling cascades was performed. In this part of the work, we focused our attention on caveolin since it modulates the activity of eNOS, ERK and Akt that are major effectors acting downstream VEGF stimulation. We demonstrated the paradoxical role of caveolin-1 preventing signaling in basal conditions and ensuring the coupling between VEGFR2 and the downstream cascades upon VEGF stimulation. We used mice deficient for the caveolin-1 gene (Cav-/-) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav+/+ mice, Cav-/- mice fails to recover a functional vasculature and actually loose part of the ligated limbs. We also isolated endothelial cells (ECs) from Cav-/- aorta and showed that on VEGF stimulation, endothelial tube formation is dramatically abrogated when compared with Cav +/+ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav-/- ECs. Interestingly, caveolin transfection in Cav-/- ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin are reached, VEGF exposure fails to activate ERK and eNOS. Altogether, these data identify caveolin as a new therapeutic target to alter VEGF signaling, in particular the cascades leading to angiogenesis and resistance to stresses.

Positron Emission Tomography, PET, is a noninvasive medical imaging technique to get functional information of the kinetics of radioactive compound injected in the body. The data used in this thesis comes from a total of five H215O PET studies of one patient. This was done in order to study the blood flow in liver metastasis of the patient, before and after treatment. A one compartment model was used to do the ROI based analyses. With a least square method in Matlab the unknown parameters in the model, such as the kinetic rate constants, the dispersion and the fraction of blood in the tissue, was extracted. Also a brief analysis of different parts of the metastases, edge and center, was done to see the variations within the metastases. The results show some increase of the blood flow after the treatment, and two of the three studied metastases showed a distinct difference of the activity in the center versus the edge. Given in the thesis are also some basic PET and compartmental modeling theory. / Positronemissionstomografi, PET, är en icke-invasiv medicinsk bildteknik för att få funktionell information om kinetiken av radioaktiva föreningar injicerade i kroppen. Det data som används i denna kandidatuppsats kommer från totalt fem H215O PET-studier av en patient. Detta gjordes för att studera blodflödet i levermetastaser hos patienten före och efter behandling. En 1-kompartmentmodell användes för att göra ROI-baserade analyser. Med en minsta kvadrat-metod i Matlab kunde de okända parametrarna i modellen, såsom den kinetiska hastighetskonstanten, spridningen och andelen blod i vävnaden, fås ut. En kort analys gjordes också av olika delar av metastaserna, kanten och mitten, för att se variationerna inuti metastaserna. Resultaten visar en viss ökning av blodflödet efter behandlingen, och två av de tre studerade metastaser visade en tydlig skillnad av aktiviteten i mitten jämfört med kanten. I rapporten ges också grundläggande teori om bland annat PET och kompartmentmodellering.