Search

How changes in bone homeostasis affect immune development is not fully understood. The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor (HIF) degradation, which is involved in cellular adaptation to low oxygen environments. Conditional deletion of Vhl in osteoblasts and hematopoietic progenitors have demonstrated a role for VHL in these cell types. Studies have demonstrated that the B cell development is mediated by crosstalk between the skeletal and hematopoietic systems. To understand how changes in bone homeostasis may affect immune cell development, we utilized Dmp1-Cre;Vhlfl/fl conditional knockout mice (VhlcKO), in which Vhl is deleted primarily in osteocytes, mature osteoblasts, and a small subset of MSCs. The VhlcKO mice display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and an overall decrease in BM cellularity compared to wild-type (WT) controls. In line with this, the frequencies, and numbers of B cells in the BM were significantly decreased. These data suggest that changes in bone homeostasis may adversely affect B cell development in a cell-extrinsic manner. We hypothesized that normal B cell development was not supported in VhlcKO mice due to alterations of the microenvironment niche, such as reduction of key niche cells and decreased production of B cell-supporting cytokines. Furthermore, we have obtained evidence that Vhl deletion also affects myeloid and erythroid development. We found elevated Epo levels in cKO peripheral blood serum and BM fluid by 6 weeks of age, and evidence for dysregulated erythropoiesis. Moreover, the cKO displayed an increased frequency of common myeloid progenitors, CD11b+ Gr1- monocytes, and CD11b+ Gr1+ granulocytes by 10 weeks of age. We also hypothesized that alterations in skeletal glucose metabolism directly affect myeloerythroid development in the BM through increased Epo-receptor (EpoR) signaling. EpoR is expressed on osteoprogenitors, hematopoietic stem cells, and B cells, but whether it is expressed on myeloid progenitors is unclear. Studies are in progress to test the hypothesis that overproduction of Epo by Vhl-deficient Dmp1+ cells result in elevated EpoR signaling in myeloid progenitors and lineages; this in turn, results in an increase in myeloid cell glycolysis and Glut1 expression. These studies are relevant for the understanding how BM microenvironmental changes can dysregulate B cell development and trigger adaptations in immunometabolism of myeloid cells. My thesis works expands on the potential mechanisms by which VHL in Dmp1-expressing cells regulate the development of distinct hematopoietic cell lineages in the BM, further elucidating and expanding our definition of “immune niches” to include osteocytes, bone, and vasculature.

Metastatic Renal Cell Carcinoma(RCC) has limited safe and effective treatments available. This is due to a lack of understanding relating to the mechanism of distant cancer metastasis. Metastatic crosstalk occurs between pseudo-hypoxic von Hippel-Lindau(VHL) knockout tumor cells and VHL wildtype tumor cells. VHL knockout cells have increased production of exosomes due to their pseudo-hypoxic state to help cells adapt to hypoxic conditions. VHL knockout-derived exosomes increase the metastatic properties of VHL wildtype recipient cells. Metastatic properties such as migration and metastasis occur in response between isolated exosomes from the CRISPR modified VHL(-) Renca murine line and VHL(+) cells, generating a model of RCC metastasis and the potential to develop targeted diagnostics and therapeutics for metastatic disease.

How changes in bone homeostasis affect immune development is not fully understood. The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor (HIF) degradation, which is involved in cellular adaptation to low oxygen environments. Conditional deletion of Vhl in osteoblasts and hematopoietic progenitors have demonstrated a role for VHL in these cell types. Studies have demonstrated that the B cell development is mediated by crosstalk between the skeletal and hematopoietic systems. To understand how changes in bone homeostasis may affect immune cell development, we utilized Dmp1-Cre;Vhlfl/fl conditional knockout mice (VhlcKO), in which Vhl is deleted primarily in osteocytes, mature osteoblasts, and a small subset of MSCs. The VhlcKO mice display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and an overall decrease in BM cellularity compared to wild-type (WT) controls. In line with this, the frequencies, and numbers of B cells in the BM were significantly decreased. These data suggest that changes in bone homeostasis may adversely affect B cell development in a cell-extrinsic manner. We hypothesized that normal B cell development was not supported in VhlcKO mice due to alterations of the microenvironment niche, such as reduction of key niche cells and decreased production of B cell-supporting cytokines. Furthermore, we have obtained evidence that Vhl deletion also affects myeloid and erythroid development. We found elevated Epo levels in cKO peripheral blood serum and BM fluid by 6 weeks of age, and evidence for dysregulated erythropoiesis. Moreover, the cKO displayed an increased frequency of common myeloid progenitors, CD11b+ Gr1- monocytes, and CD11b+ Gr1+ granulocytes by 10 weeks of age. We also hypothesized that alterations in skeletal glucose metabolism directly affect myeloerythroid development in the BM through increased Epo-receptor (EpoR) signaling. EpoR is expressed on osteoprogenitors, hematopoietic stem cel

In this review article, we aimed to analyze the available data on pheochromocytomas and paragangliomas as it pertains to their not as well-recognized association with significant glycemic abnormalities in the preoperative, perioperative, and postoperative settings as well as how they should be managed clinically. Pheochromocytomas are rare adrenal tumors that account for about 0.1% of hypertension. Paragangliomas, on the other hand, are even less common and have fewer clinical manifestations. Both types of tumors may have unusual modes of presentation which can challenge even the most experienced clinicians and are easy to overlook, resulting in post-mortem diagnosis. We wish to draw further attention to the life-threatening effects on glucose and insulin homeostasis that can occur in the form of hyperglycemic and hypoglycemic states. Hyperglycemia is a result of a glucose intolerant state created in the setting of catecholamine excess, which can present in the form of resistant diabetes, diabetic ketoacidosis (DKA), or even hyperglycemic hyperosmolar states (HHS). In many reported cases, these abnormalities resolve with resection of the tumor. However, past clinicians have also described a state of "reactive hypoglycemia" that can occur following tumor resection, further emphasizing the need for very close perioperative and postoperative monitoring. Severe hypoglycemia may also occur with inherited diseases linked to pheochromocytoma such as von Hippel-Lindau (VHL) disease as well as predominantly epinephrine-producing tumors, given some of the dramatic downstream effects of alpha and beta adrenoceptor agonization. While much of the data remains anecdotal, clinicians will benefit from the awareness of the protean manifestations of these tumors and the varied and lesser-known effects on glucose and insulin homeostasis.

The hypoxia-inducible factor 1a (HIF-1a) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1a knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHLmutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1a in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1a-positive and HIF-1a-negative, tumor cell population. Thus, activation of HIF-1a is likely an early event in VHLdefective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1a/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.

Die ständige Verfügbarkeit von molekularem Sauerstoff (O2) ist ein elementarer Bestandteil multizellulärer Lebensformen. Zur Aufrechterhaltung der Homöostase sind diese auf die Bildung des Energiesubstrates ATP durch oxidative Phosphorylierung angewiesen. Aus diesem Grunde mußten höhere Organismen während der Evolution komplexe Systeme entwickeln, die die Aufnahme und Verteilung von O2 in jede Zelle sicherstellen, sowie eine Adaptation in Phasen der Hypoxie erlauben. Mit der Identifikation des Transkriptionsfaktors "Hypoxia-inducible Factor-1" (HIF-1, 1995) wurde ein entscheidender Regulator der hypoxischen Adaptation gefunden. Unter anderem werden Prozesse wie die Erythropoiese, die Angiogenese, die Modulation des Gefäßtonus, des Glukosetransportes und der Glykolyse wesentlich durch HIF reguliert. HIF ist ein Heterodimer bestehend aus zwei Untereinheiten; einer konstitutiven beta- und einer regulativen alpha-Untereinheit. Letztere zeigt ein inverses Expressionsmuster zur perizellulären O2-Konzentration. Unter normoxischen Bedingungen ist HIFalpha instabil und wird mit einer Halbwertzeit von nur wenigen Minuten degradiert. Erst unter Hypoxie wird HIFalpha stabilisiert und ist transkriptionell aktiv. Es konnten bisher zwei funktionell relevante O2-abhängige alpha-Untereinheiten identifiziert werden: HIF-1 und HIF-2alpha. Die Bedeutung dieser beiden Systeme, der unterliegenden Regulationsmechanismen sowie die Relevanz dieses Systems in vivo waren weitgehend ungeklärt und sind wesentlicher Teil der hier zusammengefaßten Arbeiten. In den vorgelegten Studien ist es gelungen, die Expression und Regulation der beiden unterschiedlichen HIFalpha Isoformen sowohl in der Zellkultur, als auch in gesunden Geweben zu charakterisieren. In Zellkulturen zeigte sich ein sehr ähnliches Regulationsmuster hinsichtlich der O2-abhängigen Degradation, bzw. dem Induktionsverhalten unter Hypoxie, sowie der chemisch/pharmakologischen Modulation, so dass offensichtlich beide Isoformen über den, The permanent availability of molecular oxygen (O2) is an elemental need of multicellular life. For the maintenance of hemeostasis these are dependent on generation of the energy substrate ATP by oxidative phoshorylation. For this reason higher organisms had to develop complex systems during evolution that ensure the uptake and distribution of O2 into each cell, as well as permit adaptation to phases of hypoxia. With the identification of the transcription factor "Hypoxia-inducible Factor-1" (HIF-1, 1995) a master regulator of hypoxic adaptation has been found. Amongst others processes like erythropoiesis, angiogenesis, modulation of vascular tone, glucose transport and glycolysis are largely regulated by HIF. HIF is a heterodimer consisting of two subunits, a constitutive beta and a regulative alpha subunit. The latter shows an inverse relationship to the pericellular O2 concentration. HIFalpha is instable under normoxic conditions and degrades with a half life of only a few minutes. Under hypoxia the HIFalpha subunits are stabilised and are transcriptionally active. To date two functionally relevant O2-dependent alpha subunits have been identified: HIF-1 and HIF-2alpha. The importance of these two systems, the underlying regulatory mechanisms, as well as the relevance of this system in vivo were largely unknown and are a major part of the summarised studies. The presented work succeeded in characterising the expression and regulation of both HIFalpha isoforms in cell culture as well as healthy tissues. In tissue culture a very similar pattern of regulation was seen for oxygen dependent degradation, induction under hypoxia and chemical/pharmacological modulation, indicating that both subunits are regulated by the same O2-sensing and transduction apparatus. We undertook a systematic analysis of expression and regulation of both HIFalpha subunits in tissues of healthy rats. Signals for HIFalpha could only be seen under systemic hypoxia. Interestingly, both subunits w

Die ständige Verfügbarkeit von molekularem Sauerstoff (O2) ist ein elementarer Bestandteil multizellulärer Lebensformen. Zur Aufrechterhaltung der Homöostase sind diese auf die Bildung des Energiesubstrates ATP durch oxidative Phosphorylierung angewiesen. Aus diesem Grunde mußten höhere Organismen während der Evolution komplexe Systeme entwickeln, die die Aufnahme und Verteilung von O2 in jede Zelle sicherstellen, sowie eine Adaptation in Phasen der Hypoxie erlauben. Mit der Identifikation des Transkriptionsfaktors "Hypoxia-inducible Factor-1" (HIF-1, 1995) wurde ein entscheidender Regulator der hypoxischen Adaptation gefunden. Unter anderem werden Prozesse wie die Erythropoiese, die Angiogenese, die Modulation des Gefäßtonus, des Glukosetransportes und der Glykolyse wesentlich durch HIF reguliert. HIF ist ein Heterodimer bestehend aus zwei Untereinheiten; einer konstitutiven beta- und einer regulativen alpha-Untereinheit. Letztere zeigt ein inverses Expressionsmuster zur perizellulären O2-Konzentration. Unter normoxischen Bedingungen ist HIFalpha instabil und wird mit einer Halbwertzeit von nur wenigen Minuten degradiert. Erst unter Hypoxie wird HIFalpha stabilisiert und ist transkriptionell aktiv. Es konnten bisher zwei funktionell relevante O2-abhängige alpha-Untereinheiten identifiziert werden: HIF-1 und HIF-2alpha. Die Bedeutung dieser beiden Systeme, der unterliegenden Regulationsmechanismen sowie die Relevanz dieses Systems in vivo waren weitgehend ungeklärt und sind wesentlicher Teil der hier zusammengefaßten Arbeiten. In den vorgelegten Studien ist es gelungen, die Expression und Regulation der beiden unterschiedlichen HIFalpha Isoformen sowohl in der Zellkultur, als auch in gesunden Geweben zu charakterisieren. In Zellkulturen zeigte sich ein sehr ähnliches Regulationsmuster hinsichtlich der O2-abhängigen Degradation, bzw. dem Induktionsverhalten unter Hypoxie, sowie der chemisch/pharmakologischen Modulation, so dass offensichtlich beide Isoformen über den, The permanent availability of molecular oxygen (O2) is an elemental need of multicellular life. For the maintenance of hemeostasis these are dependent on generation of the energy substrate ATP by oxidative phoshorylation. For this reason higher organisms had to develop complex systems during evolution that ensure the uptake and distribution of O2 into each cell, as well as permit adaptation to phases of hypoxia. With the identification of the transcription factor "Hypoxia-inducible Factor-1" (HIF-1, 1995) a master regulator of hypoxic adaptation has been found. Amongst others processes like erythropoiesis, angiogenesis, modulation of vascular tone, glucose transport and glycolysis are largely regulated by HIF. HIF is a heterodimer consisting of two subunits, a constitutive beta and a regulative alpha subunit. The latter shows an inverse relationship to the pericellular O2 concentration. HIFalpha is instable under normoxic conditions and degrades with a half life of only a few minutes. Under hypoxia the HIFalpha subunits are stabilised and are transcriptionally active. To date two functionally relevant O2-dependent alpha subunits have been identified: HIF-1 and HIF-2alpha. The importance of these two systems, the underlying regulatory mechanisms, as well as the relevance of this system in vivo were largely unknown and are a major part of the summarised studies. The presented work succeeded in characterising the expression and regulation of both HIFalpha isoforms in cell culture as well as healthy tissues. In tissue culture a very similar pattern of regulation was seen for oxygen dependent degradation, induction under hypoxia and chemical/pharmacological modulation, indicating that both subunits are regulated by the same O2-sensing and transduction apparatus. We undertook a systematic analysis of expression and regulation of both HIFalpha subunits in tissues of healthy rats. Signals for HIFalpha could only be seen under systemic hypoxia. Interestingly, both subunits w