The research in this thesis investigated the role of the hypoxia-inducible factor (HIF) family of transcription factors in metabolism and cardiopulmonary physiology. Specifically, the effects of HIF on ventilatory control, carotid body morphology, and cardiac metabolism and function were studied using a murine model of a genetic disorder of oxygen sensing known as Chuvash polycythaemia. HIF coordinates oxygen-regulated gene expression throughout all organ systems, thereby orchestrating cellular, tissue and systemic responses to hypoxia. HIF is primarily regulated by oxygen-dependent prolyl hydroxylase-domain enzymes (PHDs) that initiate its degradation via the von Hippel-Lindau protein (VHL). In Chuvash polycythaemia, a homozygous VHL mutation in humans causes generalised stabilisation of HIF in euoxia, resulting in profound changes in cardiopulmonary physiology, exercise and metabolism. The Chuvash mouse model provides an opportunity to further characterise the role of HIF in different organ systems. Chapter 2 of this thesis introduces the murine model, demonstrating an increase in haemoglobin and haematocrit in the Chuvash mice as well as a marked reduction in body weight. Chapter 3 describes the ventilatory and carotid body study. Chuvash mice were shown to have elevated baseline ventilation in euoxia and marked ventilatory sensitivity to hypoxia. These findings were accompanied by changes within the carotid body, including hyperplasia, hypertrophy and altered ultrastructure of the oxygen-sensing type I cells. Chapter 4 of this thesis describes the study into cardiac metabolism, energetics and function. Chuvash hearts were found to have increased glycolytic flux and lactate production (the latter both in and ex vivo), with altered myocardial energetics. Despite this, left ventricular function remained normal, although in vivo cine MRI revealed clear evidence of pulmonary hypertension and right ventricular hypertrophy. Overall, this thesis provides evidence that the PHD-VHL-HIF axis plays a major role in calibrating the hypoxic response in the principal organ systems responsible for oxygen uptake, delivery and utilisation.

<p>The carotid body (CB) is a major arterial chemoreceptor containing glomus cells that are activated by changes in arterial blood contents including oxygen. Despite significant advancement in the characterization of their physiological properties, our understanding on the underlying molecular machinery and signaling pathway in CB glomus cells is still limited. </p><p>To overcome these limitations, in chapter 1, I demonstrated the first transcriptome profile of CB glomus cells using single cell sequencing technology, which allowed us to uncover a set of abundantly expressed genes, including novel glomus cell-specific transcripts. These results revealed involvement of G protein-coupled receptor (GPCR) signaling pathway, various types of ion channels, as well as atypical mitochondrial subunits in CB function. I also identified ligands for the mostly highly expressed GPCR (Olfr78) in CB glomus cells and examined this receptor’s role in CB mediated hypoxic ventilatory response. </p><p>Current knowledge of CB suggest glomus cells rely on unusual mitochondria for their sensitivity to hypoxia. I previously identified the atypical mitochondrial subunit Ndufa4l2 as a highly over-represented gene in CB glomus cells. In chapter 2, to investigate the functional significance of Ndufa4l2 in CB function, I phenotyped both Ndufa4l2 knockout mice and mice with conditional Ndufa4l2 deletion in CB glomus cells. I found that Ndufa4l2 is essential to the establishment of regular breathing after birth. Ablating Ndufa4l2 in postnatal CB glomus cells resulted in defective CB sensitivity to hypoxia as well as CB mediated hypoxic ventilatory response. Together, our data showed that Ndufa4l2 is critical to respiratory control and the oxygen sensitivity of CB glomus cells.</p> / Dissertation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O aumento de doenças cardiovasculares possui como fator primordial e, mais comum, o aumento da pressão arterial (PA). Esta, pode gerar complicações em outros órgãos e acarretar diversas patologias, podendo levar a morte. A hipertensão é uma síndrome multifatorial, cuja maior incidência ocorre em indivíduos obesos, sedentários e consumidores em excesso de bebidas alcoólicas e sal. O corpo carotídeo (CC) é um órgão quimiorreceptor localizado na bifurcação da artéria carótida, formado de estruturas básicas chamadas glomus. Cada glomus carotídeo é constituído de células tipo 1 envoltas por células tipo 2 ou sustentaculares. Este trabalho teve como objetivo analisar as alterações morfofuncionais que ocorrem no CC, causada por hipertensão arterial induzida pelo L-NAME, um inibidor da enzima óxido nítrico sintase. Para isso, o estudo utilizou 20 ratos Wistar divididos em dois grupos: controle (C) e L-NAME (LN). Após a administração de 40mg/kg/dia de L-NAME por 45 dias, o CC foi coletado. Observou-se aumento significativo da pressão arterial a partir da segunda semana de administração de L-NAME. Análise quantitativa mostrou uma redução no número de núcleos do glomus carotídeo e o aumento na área total do órgão no grupo LN. Não foi encontrado diferença significativa no número de núcleos totais do corpo carotídeo entre os grupos. Na análise morfológica do grupo LN, observamos a formação de vacúolos nas células tipo 1 do glomus, bem como uma redução do número total de núcleos das células de cada glomus carotídeo. A análise qualitativa sugeriu um aumento no número de fibras colágenas e fibras do sistema elástico na matriz extracelular e grânulos no grupo LN. Imunomarcações com anticorpo anti VEGF e NF-kB e nNOS mostram-se aumentadas e dispersas por todo CC no grupo LN em relação ao grupo C. Além disso, marcações para Substância-P também foram observadas em maior quantidade nas células tipo 1 do grupo LN. Quanto à marcação para PGP 9.5, houve a redução desta marcação caracterizada dentro do glomus carotídeo grupo LN comparado ao grupo C. O estudo sugere que o corpo carotídeo, em resposta à hipertensão induzida pela inibição da enzima óxido nítrico sintase, gera mudanças morfofisiológicas semelhantes as encontradas em hipóxia. / The increased cardiovascular disease as the key features and, more common, increasing blood pressure (BP). This can cause complications in other organs and cause various diseases and may lead to death. Hypertension is a multifactorial syndrome, whose incidence is higher in obese, sedentary and consumers in excess alcohol and salt. The carotid body (CC) is a chemoreceptor organ located in bifurcation carotid artery, consisting of basic structures called carotid glomus. Each is made up of carotid glomus type 1 cells surrounded by sustentacular or type 2 cells. This study aimed to analyze the morphological and functional changes that occur in CC, caused by hypertension induced by L-NAME, an inhibitor of nitric oxide synthase. For that, the study used 20 rats divided into two groups: control (C) and L-NAME (LN). After administration of 40mg/kg/day L-NAME for 45 days, the CC was collected. We observed significantly increased blood pressure starting at second week of administration of L-NAME. Quantitative analysis showed a reduction in the number of cells in the carotid glomus and an increase in the total area of the body in the LN group. No significant difference was found in the nuclei total number in carotid body between the groups. In the morphological analysis of the LN group, we observed the formation of vacuoles in type 1 glomus cells, as well as a reduction in the total number of nuclei of cells from each carotid glomus. The qualitative analysis suggested an increase in collagen and elastic system fibers in the extracellular matrix and cytoplasmic granules in LN group. Immunostainings with anti-VEGF and NFkappa- B and nNOS show was increase and dispersed in the LN group than in group C. In addition, appointments for P substance were also obeservado in greater amounts in type 1 cells of the LN group. The marking for PGP 9.5, there was a reduction of this marking characterized in carotid glomus LN group compared with group C. The study suggests that the carotid body, in response to hypertension induced by inhibition of nitric oxide synthase, produces morphophysiological changes similar to those found in hypoxia.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Oxygen-sensing cells release neurotransmitters, including serotonin, in response to hypoxia in the blood or surrounding air/water. This stimulates the glossopharyngeal and/or vagal nerves, triggering increased ventilation via the respiratory reflex. In the adult, they are located in the carotid body (glomus cells) and lung epithelia (pulmonary neuroendocrine cells) of amniotes, and in the epithelia of the gills and orobranchial cavity (‘neuroepithelial cells’) of anamniotes. Despite their physiological importance, little is known about the molecular mechanisms of their development, while the evolutionary relationships between the various oxygen-sensing cell types are unknown. The chromaffin cells of the mammalian adrenal medulla are hypoxia-sensitive transiently during neonatal life. Both carotid body glomus cells and adrenal chromaffin cells arise from the neural crest and require the transcription factors Phox2b and Ascl1 for their development. Given these similarities, I aimed to test the hypothesis that the same molecular mechanisms underlie their development. Expression analysis of 13 sympathoadrenal pathway genes throughout chicken carotid body development revealed striking similarities with adrenal chromaffin cell development. Analysis of mouse mutants showed that the transcription factors Hand2, Sox4 and Sox11 are required for carotid body development. In addition, loss of the receptor tyrosine kinase Ret or the transcription factor AP-2β, which significantly affects sympathetic ganglion but not adrenal chromaffin cell development, has no effect on the carotid body. Adrenal chromaffin cells differentiate from neurons that migrate into the adrenal gland from ‘primary’ sympathetic ganglia at the dorsal aorta. Carotid body glomus cells were previously proposed to arise from neuronal “émigrés” from neighbouring ganglia: the superior cervical ganglion in mammals and the nodose ganglion in the chick. However, nodose neurons are considered to be nodose placode-derived. Using electroporation and grafting in the chick, I confirmed that the nodose placode does not contribute to the carotid body, identified a small population of autonomic neural crest-derived neurons in the nodose ganglion, and confirmed the existence of “bridges” of neurons between the nodose ganglion and the carotid body. My data suggest that, like adrenal chromaffin cells, carotid body glomus cells differentiate from autonomic neural crest-derived neurons in nearby ganglia, which migrate into the carotid body primordium and down-regulate neuronal markers. The proposed evolutionary relationship between the carotid body glomus cells and the serotonin-positive neuroepithelial cells of anamniote gills has never been tested. Using vital dye labelling, neural fold grafts, genetic lineage-tracing in zebrafish and analysis of zebrafish mutants lacking all neural crest cells, I found that serotonin-positive cells in the gills and orobranchial epithelia of lamprey (jawless fish), zebrafish (ray-finned bony fish) and frog (anamniote tetrapod) are not neural crest-derived, and hence are not homologous to carotid body glomus cells. Genetic lineage-tracing in mouse and neural fold grafts in chick also confirmed that serotonin-positive neuroendocrine cells in the lung are not neural crest- derived, hence must have an endodermal origin (since the lungs are out-pocketings of the gut). My results suggest that the neuroepithelial cells of anamniotes are not related to carotid body glomus cells, but rather are homologous to the oxygen-sensing cells of the lung. Consistent with this hypothesis, I found that many genes expressed during carotid body development are not expressed by the epithelia of either chick lungs or lamprey gills. Taken together, my data suggest that as air-breathing evolved, gut endoderm- derived cells that originally responded to hypoxia in water were maintained in the lungs to monitor oxygen levels in air, while a population of neural crest-derived chromaffin cells near the pharyngeal arch arteries was recruited to monitor oxygen levels in blood.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O aumento de doenças cardiovasculares possui como fator primordial e, mais comum, o aumento da pressão arterial (PA). Esta, pode gerar complicações em outros órgãos e acarretar diversas patologias, podendo levar a morte. A hipertensão é uma síndrome multifatorial, cuja maior incidência ocorre em indivíduos obesos, sedentários e consumidores em excesso de bebidas alcoólicas e sal. O corpo carotídeo (CC) é um órgão quimiorreceptor localizado na bifurcação da artéria carótida, formado de estruturas básicas chamadas glomus. Cada glomus carotídeo é constituído de células tipo 1 envoltas por células tipo 2 ou sustentaculares. Este trabalho teve como objetivo analisar as alterações morfofuncionais que ocorrem no CC, causada por hipertensão arterial induzida pelo L-NAME, um inibidor da enzima óxido nítrico sintase. Para isso, o estudo utilizou 20 ratos Wistar divididos em dois grupos: controle (C) e L-NAME (LN). Após a administração de 40mg/kg/dia de L-NAME por 45 dias, o CC foi coletado. Observou-se aumento significativo da pressão arterial a partir da segunda semana de administração de L-NAME. Análise quantitativa mostrou uma redução no número de núcleos do glomus carotídeo e o aumento na área total do órgão no grupo LN. Não foi encontrado diferença significativa no número de núcleos totais do corpo carotídeo entre os grupos. Na análise morfológica do grupo LN, observamos a formação de vacúolos nas células tipo 1 do glomus, bem como uma redução do número total de núcleos das células de cada glomus carotídeo. A análise qualitativa sugeriu um aumento no número de fibras colágenas e fibras do sistema elástico na matriz extracelular e grânulos no grupo LN. Imunomarcações com anticorpo anti VEGF e NF-kB e nNOS mostram-se aumentadas e dispersas por todo CC no grupo LN em relação ao grupo C. Além disso, marcações para Substância-P também foram observadas em maior quantidade nas células tipo 1 do grupo LN. Quanto à marcação para PGP 9.5, houve a redução desta marcação caracterizada dentro do glomus carotídeo grupo LN comparado ao grupo C. O estudo sugere que o corpo carotídeo, em resposta à hipertensão induzida pela inibição da enzima óxido nítrico sintase, gera mudanças morfofisiológicas semelhantes as encontradas em hipóxia. / The increased cardiovascular disease as the key features and, more common, increasing blood pressure (BP). This can cause complications in other organs and cause various diseases and may lead to death. Hypertension is a multifactorial syndrome, whose incidence is higher in obese, sedentary and consumers in excess alcohol and salt. The carotid body (CC) is a chemoreceptor organ located in bifurcation carotid artery, consisting of basic structures called carotid glomus. Each is made up of carotid glomus type 1 cells surrounded by sustentacular or type 2 cells. This study aimed to analyze the morphological and functional changes that occur in CC, caused by hypertension induced by L-NAME, an inhibitor of nitric oxide synthase. For that, the study used 20 rats divided into two groups: control (C) and L-NAME (LN). After administration of 40mg/kg/day L-NAME for 45 days, the CC was collected. We observed significantly increased blood pressure starting at second week of administration of L-NAME. Quantitative analysis showed a reduction in the number of cells in the carotid glomus and an increase in the total area of the body in the LN group. No significant difference was found in the nuclei total number in carotid body between the groups. In the morphological analysis of the LN group, we observed the formation of vacuoles in type 1 glomus cells, as well as a reduction in the total number of nuclei of cells from each carotid glomus. The qualitative analysis suggested an increase in collagen and elastic system fibers in the extracellular matrix and cytoplasmic granules in LN group. Immunostainings with anti-VEGF and NFkappa- B and nNOS show was increase and dispersed in the LN group than in group C. In addition, appointments for P substance were also obeservado in greater amounts in type 1 cells of the LN group. The marking for PGP 9.5, there was a reduction of this marking characterized in carotid glomus LN group compared with group C. The study suggests that the carotid body, in response to hypertension induced by inhibition of nitric oxide synthase, produces morphophysiological changes similar to those found in hypoxia.

Background: Carotid body tumor (CBT) is a rare lesion of the neuroendocrine system. Chronic hypoxia has long been recognized as an etiology of CBT and other paragangliomas. Recent biogenetic discoveries reveal that mutations in oxygen-sensing genes are another etiology, accounting for approximately 35% of cases, and that these 2 etiologies are probably additive. Data Sources: (1) A retrospective analysis of fifteen cases of CBT in a 6-year period occurring in the mountains of Southern Appalachia; (2) an extensive review of the literature on the surgery of CBT and on the expansive biogenetic understanding of the disease. Conclusions: Improved imaging, vascular surgical techniques, and understanding of the disease have vastly improved outcomes for patients. The necessities for long-term follow-up and appropriate genetic testing and counseling of patients and their families are documented. Surgeon and institutional competence are critical in achieving maximal outcomes.

The major physiological function of the carotid body is to respond to a low partial pressure of oxygen in the systemic arterial blood. The structure and functions of the adult carotid body have been extensively investigated over the past fifteen years. However, the carotid body in children has been relatively neglected with only a handful of studies being performed. To date, no study has been undertaken to investigate the effects of intrauterine hypoxia on the carotid body of foetuses. Clinically, intrauterine growth retardation has been ascribed, amongst other causes, to placental insufficiency that results in chronic hypoxia in the fetus. Intrauterine growth retardation can be divided into two types: - Type I (symmetrical) and type II (asymmetrical). In Type II intrauterine growth retardation, growth retardation does not become clinically evident until the third trimester. There is relative brain sparing with a greater deprivation in the size of abdominal organs, such as the liver and the kidneys. Previous studies have shown that there is no correlation between volume of the carotid body and hypoxia in children. However, Heath et al. made the observation that there are three variants of chief cells (progenitor, light and dark) within the carotid body and that an increase in the relative percentage of the dark subtype is an indicator of hypoxia. Using this observation, the present study set out to test two hypotheses: Firstly, whether the carotid body is functional in utero; and secondly whether there are any objective morphological changes in the carotid bodies of fetuses that have been subjected to intrauterine growth retardation. The carotid bodies from 72 fetuses with a gestational age between thirty and forty weeks were removed from the archived autopsy material, and differential cell counts were performed of the various cells present within the carotid bodies, using haematoxylin and eosin stained sections of the carotid bodies. The cases were assigned to three groups: - I) cases that had clinical and pathological evidence of intrauterine growth retardation, 2) negative controls and 3) positive controls. The three main groups were categorised as follows: -: (1) Intrauterine growth retardation (all cases with a weight for gestational age that is below the tenth centile and a brain to liver ratio of greater than four.) (2) Negative controls (all cases in whom there is a normal weight for age, a brain to liver ratio of less than three and no histological evidence of an episode of significant hypoxia before death). (3) Positive controls (all cases in whom there was clinically significant hypoxia present before death). The groups comprised of: 20 hypoxic positive controls, 15 negative controls, and 16 test cases which had suffered from intrauterine growth retardation. The remaining 21 cases were 7 dysmorphic infants, 3 congenital infection cases (congenital syphilis) and 11 cases that fitted the negative control criteria but had suffered significant hypoxia, thus excluding them from that category. The results showed that no significant difference was present in the percentage of sustentacular cells between any of the three groups. The results of the percentage of dark chief cells were as follows: l) mean percentage of dark chief cells in the intrauterine growth retardation group was 21.1 ±10.9%. 2) mean percentage of dark chief cells in the negative controls was 12.3 ±7.3%. 3) mean percentage of dark chief cells in the positive controls was 21.2 ±9.8%. A significant difference was present between the intrauterine growth retardation cases and the negative controls p=0.013, and between the positive and negative controls p=0.006. The dark chief cell count in the intrauterine growth retardation group showed no significant difference from the positive controls. No age-related difference appeared to be present in any of the groups. The conclusions reached are: a) Clinical hypoxia correlates with morphological changes in the carotid body, manifesting as an increase in the percentage of dark chief cells. b) intrauterine growth retardation cases show similar morphological changes in the carotid body to cases that have suffered from clinical hypoxia. c) therefore, by deduction intrauterine growth retardation fetuses have probably also been exposed to significant hypoxia while in utero. d) the fact that morphological changes in response to hypoxia are occurring in the carotid bodies of fetuses is an indication that the carotid body may be functional in utero. The results of the study indicate that a dark chief cell percentage of greater than 20% indicates that the fetus has been subjected to significant hypoxia, while a percentage of less than 10% indicates that it has not. A percentage of between 10 and 20% is unhelpful in determining whether hypoxia has taken place. The results of this study indicate that histological examination of the carotid bodies in neonates suspected of intrauterine growth retardation could be a useful additional means of assessment.

Carotid body (CB) receptor type I cells transduce blood-borne chemical stimuli into electrical signals and release the excitatory neurotransmitter ATP onto afferent terminals that project to the breathing centre located in the brainstem. Within the CB, type I cells are ensheathed by glial-like processes of type II cells. Recently, it was hypothesized that type II cells have a paracrine function in CB chemotransduction by acting as an ATP amplifier and enhancing chemoexcitation (Zhang et al. 2012). Given this recent development, the primary goal of this thesis was to further elucidate the paracrine function of type II cells and characterize the signalling mechanisms involved in type I and type II cell interactions. Ratiometric calcium imaging was used to investigate type II cell sensitivity to two prominent CB neuromodulators, angiotensin II (ANG II) and 5-HT, in rat CB cultures. Both ANG II and 5-HT elicited large rises in intracellular Ca<sup>2+<sup> that were present in the absence of extracellular Ca<sup>2+<sup> and were inhibited by intracellular store depletion agents. ANG II and 5-HT acted on their respective G-protein coupled receptors, AT<sub>1<sub> receptor and 5-HT<sub>2A<sub> receptor, to initiate these Ca<sup>2+<sup> responses presumably via a PLC-IP<sub>3<sub> mediated mechanism. Interestingly, these Ca<sup>2+<sup> responses were required to activate pannexin-1 channels (Panx-1), a channel that has been previously shown to be a conduit for ATP in type II cells (Zhang et al. 2012). We were also interested in determining whether type II cells were capable of indirectly responding to a chemostimulus such that the stimulus would elicit neurosecretion from type I cells and result in a secondary Ca<sup>2+<sup> responses in type II cells. Isohydric hypercapnia and a depolarizing stimulus (30 mM KCl saline) were capable of eliciting indirect Ca<sup>2+<sup> responses in type II cells. These secondary Ca<sup>2+<sup> responses in type II cells were partially inhibited by suramin, a purinergic P2Y2 receptor antagonist, suggesting that ATP was the predominant neurotransmitter responsible for type I to type II crosstalk. Similarly, a selective agonist for type II cells, UTP, evoked indirect Ca<sup>2+<sup> responses in nearby type I cells. Type II to type I cell communication was dependent on Panx-1 channels since the secondary Ca<sup>2+<sup> responses in type I cells were inhibited by the Panx-1 blocker, carbenoxolone (5 µM). UTP-evoked indirect Ca<sup>2+<sup> in type I cells were partially inhibited by adenosine A<sub>2<sub> receptor antagonists suggesting that the neuromodulator, adenosine, governs cross-talk between type II and type I cells. This study elucidates the importance of purinergic signalling in the bi-directional cross-talk between receptor type I cells and glial-like type II cells. / Thesis / Master of Science (MSc)

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