Mesenchymal stromal cells (MSC) are bone marrow-derived, non-hematopoietic progenitors that are amenable to genetic engineering, making them attractive delivery vehicles for therapeutic proteins. However, limited transplanted cell survival compromises the efficacy of MSC-based gene therapy. We hypothesized that co-implantation of insulin-like growth factor-1 (IGF-I)-overexpressing MSC (MSC-IGF) would improve MSC-based therapy of anemia by providing paracrine support to erythropoietin (EPO)-secreting MSC (MSC-EPO). Murine MSC were found to express the IGF-I receptor and be responsive to IGF-I stimulation. IGF-I also improved MSC survival in vitro. MSC were admixed in a bovine collagen matrix and implanted by subcutaneous injection in a murine model of chronic renal failure. Mice receiving MSC-EPO co-implanted with MSC-IGF experienced a greater and significantly sustained elevation in hematocrit compared to controls; heart function was also improved. Co-implantation of MSC-IGF therefore represents a promising new strategy for enhancing implanted cell survival, and improving cell-based gene therapy of renal anemia.

The insulin-like growth factor (IGF) system plays an important role in a number of disease states, such as cancer, and has also been implicated in wound and burn healing processes. Two IGF receptors, the type-1 IGF and type-2 IGF receptors, as well as six insulin-like growth factor binding proteins (IGFBP-1 to 6), have well established roles in mediating IGF activity. Earlier studies in this laboratory demonstrated that IGF-II binds to the extracellular matrix (ECM) protein vitronectin (VN), and although IGF-I does not bind directly to VN it can bind indirectly via specific IGFBPs. Therefore the aim of the research described in this thesis was to determine whether binary and ternary complexes of IGF-I/II, IGFBPs and VN affect human keratinocyte cell function. The strategy of pre-binding these complexes to the culture dishes was adopted in this study in an attempt to more accurately reflect the extracellular environment in vivo. These studies demonstrated that the binary complex of IGF-II and VN and the ternary complexes comprised of IGF-I, IGFBP-2, or 3, or 4, or 5 and VN significantly stimulated HaCaT de novo cell protein synthesis in the human keratinocyte cell line. Interestingly, these latter experiments demonstrated that although large increases in protein synthesis were observed using the ternary complexes, IGF-I/IGFBP complexes alone were responsible for the significant increases in protein synthesis and these responses are mediated via the MAPK signaling pathway. In addition, both the dimeric and trimeric complexes significantly enhanced cell migration through 12 μm TranswellsTM. Unlike the protein synthesis assays, VN was critically important in these migratory responses and highlighting the important role that integrins play in cell migration. Cell attachment assays on the other hand demonstrated that the interactions of IGFs with IGFBPs and VN did not affect cell attachment. The data encompassed within this thesis represent the first studies to provide a functional role for the interaction between IGFs, IGFBPs and VN in human keratinocytes. Taken together these results suggest that IGF/IGFBP/VN complexes may hold great potential in situations where enhanced keratinocyte cell migration and proliferation is required, such as in wound healing and skin engineering applications.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 162 pages. Includes Vita. Includes bibliographical references.

A leishmaniose visceral é uma infecção grave que leva a pancitopenia. Quando se trata de disfunções medulares decorrentes de infecção por Leishmania (Leishmania) infantum há poucas abordagens descrevendo as alterações na mielopoiese e os mecanismos que levam a pancitopenia na LV. Alguns estudos demonstram uma relação importante entre a pancitopenia e o fator de crescimento insulin-like growth fator-I (IGF-I), no entanto, o seu papel endógeno na hematopoiese ainda não está claro. Propomos estudar a influência desse fator na hematopoiese e sua relação com o desenvolvimento da pancitopenia em hamsteres infectados por via intra-peritoneal com 2x107 de amastigotas por L. (L.) infantum. Avaliamos em 90 e 120 dias pós-infecção a LDU (Leishman-Donovan units) no baço e fígado, quando observamos tendência à progressão da infecção. Aos 60 dias pós-infecção, os animais com LV desenvolveram a plaquetopenia como primeira alteração hematológica, e a partir dos 90 dias pós-infecção, a anemia, e a leucopenia com reduções significantes nos leucócitos totais, linfócitos e neutrófilos. Já aos 120 dias de infecção, os leucócitos totais diminuíram significantemente acompanhados por uma redução de linfócitos, monócitos e eosinófilos. A partir desses dados, focamos a análise da medula óssea nos hamsteres com 90 e 120 pós-infecção. No mielograma, vimos alterações somente nos hamsteres com LV aos 90 dias pós-infecção, com um aumento significante na proporção células mielóides imaturas: células mielóides maduras. Na biópsia da tíbia, houve um aumento significante da celularidade quando comparados com seu respectivo controle, apenas no período de 90 dias pós-infecção. Em adição, observamos uma proliferação e/ou infiltrado macrofágico significante nos hamsteres com LV, mas sem diferença estatística nos períodos de 90 e 120 dias pós-infecção. Na avaliação semi-quantitativa de fibras de reticulina, somente aos 90 dias pós-infecção, observamos aumento significante nos infectados comparados aos controles, caracterizando um quadro fibrótico. Foi medida a expressão de mRNA de IGF-I por PCR em tempo real, aos 90 e 120 dias pós-infecção, onde ocorreu um aumento significante da expressão de IGF-I nos animais infectados em relação aos controles aos 90 dias. Como expostos, os hamsteres com LV apresentam alterações hematológicas como anemia, leucopenia e plaquetopenia, e ainda alterações na medula óssea como aumento da celularidade, proliferação macrofágica e fibrose acompanhados por um aumento da expressão de IGF-I. Assim podemos concluir que nossos dados indicam que o hamster se constitui num bom modelo para o estudo da patogênese da pancitopenia e das alterações medulares decorrentes da infecção por L.(L.) infantum. Neste modelo, ocorre alteração da expressão de IGF-I durante a evolução da infecção com possível papel na patogenia da pancitopenia. / Visceral leishmaniasis is a serious infection that leads to pancytopenia. When it comes to spinal cord dysfunction resulting from infection Leishmania (Leishmania) infantum there are few approaches describing the changes in myelopoiesis and mechanisms that lead to pancytopenia in LV. Some studies have shown a significant relationship between pancytopenia and insulin-like growth factor-growth factor I (IGF-I), however, their endogenous role in hematopoiesis is still not clear. We propose to study the influence of this factor in hematopoiesis and its relationship to the development of pancytopenia in hamsters infected intraperitoneally with 2x107 amastigotes by L. (L.) infantum. Evaluated at 90 and 120 days post-infection the LDU (Leishman-Donovan units) in the spleen and liver, when we observed a tendency to progression of the infection. At 60 days post-infection, animals with LV developed thrombocytopenia as the first hematologic changes, and from 90 days post-infection, anemia, and leukopenia with significant reductions in total leukocytes, lymphocytes and neutrophils. Already at 120 days of infection, total leukocytes decreased significantly accompanied by a decrease lymphocytes, monocytes and eosinophils. From these data, we focus on the analysis of the bone marrow in hamsters with 90 and 120 post-infection. The myelogram changes seen only in hamsters with LV at 90 days post-infection, with a significant increase in the proportion immature myeloid cells: mature myeloid cells. Biopsy of the tibia, there was a significant increase in cellularity compared with their respective control, only 90 days post-infection. In addition, we observed a proliferation and / or infiltration significant macrophage in hamsters with LV, but no statistical difference in the periods of 90 and 120 days post-infection. In semi-quantitative assessment of reticulin fibers, only at 90 days post-infection, we observed a significant increase in infected compared to controls, featuring a fibrous framework. MRNA expression of IGF-I was measured by real-time PCR, at 90 and 120 days post-infection, where a significant increase in IGF-I expression in infected animals compared to controls at 90 days occurred. As set out, the present hamsters VL hematological disorders such as anemia, leukopenia and thrombocytopenia, as well as changes in bone marrow cellularity increased, macrophage proliferation and fibrosis accompanied by a reduction in IGF-I expression. Thus we can conclude that our data indicate that the hamster is a good model to study the pathogenesis of pancytopenia and marrow changes resulting from infection by L. (L.) Infantum. In this model, alteration of IGF-I expression during the course of infection with a possible role in the pathogenesis of pancytopenia.

Abstract A hypothesis is created that an association between androgenetic alopecia (AGA) and serious cardiovascular events, such as myocardial infarction and fatal ischaemic heart disease has been reported, but the mechanism explaining this association has remained unclear. The aim of this study was to analyze the relationship between insulin resistance, (coronary) artery disease and AGA. Moreover, a hypothesis on the role of electromagnetic cell adhesion in the development of AGA is presented. In the present series of men aged 19–50 years (n = 154) with early (<  35 years of age), significant AGA of at least grade 3 (vertex) in the Hamilton classification modified by Norwood (Norwood 1975) was hyperinsulinaemia encountered twice as often as on age-matched controls. Other signs of the insulin resistance syndrome, such as obesity, lipid lowering and antihypertensive drugs were also found to correlate with early AGA. In a population-based case-control study, male patients living a small rural town who had undergone an urgent or elective coronary revascularization procedure (n =  85) and their age-matched controls were analysed after stratification by age at operation and hair status. The findings showed AGA to be more common coronary artery disease and early AGA as those with early coronary artery disease. In a population aged 63 years (n = 541, 217 men), neck circumference was found to correlate with the conventional anthropometric indicators of insulin resistance and with elevated serum insulin in both genders, which means that neck circumference is a simple anthropometric indicator of android type obesity and insulin resistance. In the same female population other factors of insulin resistance (whr, waist circumference, serum insulin level and microalbuminuria) were associated with marked (grade 2 or 3 on a modified Ludwig scale) hair loss. Paternal heredity was clearly characteristic of AGA in both genders, particularly of early AGA in men. We present a hypothesis that the overactive androgen state inhibits cell mitosis in the dermal papilla of the hair follicle and contributes to a weaker electromagnetic attraction between the undifferentiated germ cells and the dermal papilla and also to a shortened anagen phase of the hair growth cycle. Insulin resistance has an additional pathogenic role in the excessive miniaturization of the hair follicle. As a conclusion, along with android obesity, early alopecia can be considered a sign of insulin resistance and a possible risk factor for an early onset of coronary artery disease. Timely intervention in the risk factors may help to slow down or prevent the development of arterial disease and possibly also to alleviate the cosmetic and psychosocial consequences of hair loss. / Tiivistelmä Insuliiniresistenssin, (sepel)valtimotaudin ja AGA:n välillä on yhteyksiä. Taustalla olevat patomekanismit ovat kuitenkin epäselviä. Tässä väitöskirjatyössä tutkittiin insuliiniresistenssin ja (sepel)valtimotaudin suhdetta AGA:an. Lisäksi luotiin hypoteesi sähkömagneettisen soluadheesion roolista AGA:n kehittymisessä. Aineiston 19–50-vuotiailla miehillä (n = 154), joilla oli varhainen (< 35 v), merkittävä, vähintään kolmannen (vertex) asteen AGA Norwoodin modifioiman Hamiltonin luokituksen mukaan (Norwood 1975) seerumin insuliinipitoisuus oli suurentunut liki kaksi kertaa useammin kuin samanikäisillä verrokeilla. Myös muiden insuliiniresistenssioireyhtymään liitettyjen vaaratekijöiden, kuten ylipainon, havaittiin liittyvän varhaiseen AGA:an. Pienen maaseutukaupungin kaikki sepelvaltimoiden revaskularisaatioon joutuneet miehet (n = 85) analysoitiin toimenpiteeseen joutumisiän ja hiusstatuksen mukaan. Tulokset osoittavat AGA:n olevan yhteydessä sepelvaltimotautiin ja varhaisen AGA:n varhaiseen sepelvaltimotautiin. Aineiston 63-vuotiailla (n = 541, miehiä 217) kaulan ympärysmitan todettiin korreloivan selvästi antropometrisiin, insuliiniresistenssiä kuvaaviin mittoihin ja seerumin insuliinipitoisuuden kasvuun sekä miehillä että naisilla. Kaulan ympärysmitta soveltuu siten käytettäväksi antropometrisena mittana androidityyppisen ylipainon ja insuliiniresistenssin selvittämisessä. Saman väestöotoksen naisilla tehdyssä tutkimuksessa havaittiin muiden insuliiniresistenssin osatekijöiden (vyötärö-lantiosuhteen, vyötärön ympärysmitan, seerumin insuliinipitoisuuden ja mikroalbuminurian) liittyvän huomattavaan hiustenlähtöön (asteet II ja III modifioidulla Ludwigin skaalalla). AGA:ssa isän suvun perimän vaikutus oli selvä molemmilla sukupuolilla. Se oli voimakas erityisesti miesten varhaisessa AGA:ssa. Laatimamme hypoteesin mukaan suuri androgeenipitoisuus estää dermaalipapillan solujen mitoosia ja heikentää sähkömagneettista vetovoimaa. Tällöin hiusfollikkelin solujen määrää vähenee ja hiuksen kasvuvaihe lyhenee haittaavasti. Insuliiniresistenssillä on hypoteesin mukaan toissijainen rooli hiusfollikkelin pienenemisprosessissa. Aikaista hiustenlähtöä androidin ylipainon ohella voidaan pitää insuliiniresistenssin merkkinä ja riskinä sepelvaltimotaudin tavanomaista aiempaan ilmaantumiseen. Puuttumalla ajoissa vaaratekijöihin valtimotaudin kehittymistä voidaan hidastaa tai estää ja ehkä myös vähentää kosmeettisesti ja psykososiaalisesti haittaavaa hiusten menetystä.

A leishmaniose visceral é uma infecção grave que leva a pancitopenia. Quando se trata de disfunções medulares decorrentes de infecção por Leishmania (Leishmania) infantum há poucas abordagens descrevendo as alterações na mielopoiese e os mecanismos que levam a pancitopenia na LV. Alguns estudos demonstram uma relação importante entre a pancitopenia e o fator de crescimento insulin-like growth fator-I (IGF-I), no entanto, o seu papel endógeno na hematopoiese ainda não está claro. Propomos estudar a influência desse fator na hematopoiese e sua relação com o desenvolvimento da pancitopenia em hamsteres infectados por via intra-peritoneal com 2x107 de amastigotas por L. (L.) infantum. Avaliamos em 90 e 120 dias pós-infecção a LDU (Leishman-Donovan units) no baço e fígado, quando observamos tendência à progressão da infecção. Aos 60 dias pós-infecção, os animais com LV desenvolveram a plaquetopenia como primeira alteração hematológica, e a partir dos 90 dias pós-infecção, a anemia, e a leucopenia com reduções significantes nos leucócitos totais, linfócitos e neutrófilos. Já aos 120 dias de infecção, os leucócitos totais diminuíram significantemente acompanhados por uma redução de linfócitos, monócitos e eosinófilos. A partir desses dados, focamos a análise da medula óssea nos hamsteres com 90 e 120 pós-infecção. No mielograma, vimos alterações somente nos hamsteres com LV aos 90 dias pós-infecção, com um aumento significante na proporção células mielóides imaturas: células mielóides maduras. Na biópsia da tíbia, houve um aumento significante da celularidade quando comparados com seu respectivo controle, apenas no período de 90 dias pós-infecção. Em adição, observamos uma proliferação e/ou infiltrado macrofágico significante nos hamsteres com LV, mas sem diferença estatística nos períodos de 90 e 120 dias pós-infecção. Na avaliação semi-quantitativa de fibras de reticulina, somente aos 90 dias pós-infecção, observamos aumento significante nos infectados comparados aos controles, caracterizando um quadro fibrótico. Foi medida a expressão de mRNA de IGF-I por PCR em tempo real, aos 90 e 120 dias pós-infecção, onde ocorreu um aumento significante da expressão de IGF-I nos animais infectados em relação aos controles aos 90 dias. Como expostos, os hamsteres com LV apresentam alterações hematológicas como anemia, leucopenia e plaquetopenia, e ainda alterações na medula óssea como aumento da celularidade, proliferação macrofágica e fibrose acompanhados por um aumento da expressão de IGF-I. Assim podemos concluir que nossos dados indicam que o hamster se constitui num bom modelo para o estudo da patogênese da pancitopenia e das alterações medulares decorrentes da infecção por L.(L.) infantum. Neste modelo, ocorre alteração da expressão de IGF-I durante a evolução da infecção com possível papel na patogenia da pancitopenia. / Visceral leishmaniasis is a serious infection that leads to pancytopenia. When it comes to spinal cord dysfunction resulting from infection Leishmania (Leishmania) infantum there are few approaches describing the changes in myelopoiesis and mechanisms that lead to pancytopenia in LV. Some studies have shown a significant relationship between pancytopenia and insulin-like growth factor-growth factor I (IGF-I), however, their endogenous role in hematopoiesis is still not clear. We propose to study the influence of this factor in hematopoiesis and its relationship to the development of pancytopenia in hamsters infected intraperitoneally with 2x107 amastigotes by L. (L.) infantum. Evaluated at 90 and 120 days post-infection the LDU (Leishman-Donovan units) in the spleen and liver, when we observed a tendency to progression of the infection. At 60 days post-infection, animals with LV developed thrombocytopenia as the first hematologic changes, and from 90 days post-infection, anemia, and leukopenia with significant reductions in total leukocytes, lymphocytes and neutrophils. Already at 120 days of infection, total leukocytes decreased significantly accompanied by a decrease lymphocytes, monocytes and eosinophils. From these data, we focus on the analysis of the bone marrow in hamsters with 90 and 120 post-infection. The myelogram changes seen only in hamsters with LV at 90 days post-infection, with a significant increase in the proportion immature myeloid cells: mature myeloid cells. Biopsy of the tibia, there was a significant increase in cellularity compared with their respective control, only 90 days post-infection. In addition, we observed a proliferation and / or infiltration significant macrophage in hamsters with LV, but no statistical difference in the periods of 90 and 120 days post-infection. In semi-quantitative assessment of reticulin fibers, only at 90 days post-infection, we observed a significant increase in infected compared to controls, featuring a fibrous framework. MRNA expression of IGF-I was measured by real-time PCR, at 90 and 120 days post-infection, where a significant increase in IGF-I expression in infected animals compared to controls at 90 days occurred. As set out, the present hamsters VL hematological disorders such as anemia, leukopenia and thrombocytopenia, as well as changes in bone marrow cellularity increased, macrophage proliferation and fibrosis accompanied by a reduction in IGF-I expression. Thus we can conclude that our data indicate that the hamster is a good model to study the pathogenesis of pancytopenia and marrow changes resulting from infection by L. (L.) Infantum. In this model, alteration of IGF-I expression during the course of infection with a possible role in the pathogenesis of pancytopenia.

Aging can be defined as the accumulation of changes affecting the maintenance of homeostatic processes over time, leading to functional decline and increased risk for disease and death. In its simplicity, aging is the systemwide deterioration of an organism. Genetic studies have identified many potential molecular mechanisms of aging including DNA damage, telomere shortening, mitochondrial dysfunction, increased oxidative stress, uncontrolled inflammation, and hormone dysregulation (reviewed in [1]). However, in reality, aging is likely to be a combination of some (or potentially all) of these mechanisms. Interestingly, aging and metabolism are tightly coordinated. Aging is a major contributor to metabolic decline and related diseases, including type 2 diabetes, metabolic syndrome, and cancer. One of the best characterized metabolic pathways implicated in aging is the insulin/IGF-1 signaling (IIS) pathway. Downstream signaling components of the IIS pathway receptor have been well studied and include an interconnected network of signaling events that regulate many physiological outputs. However, less is known about the role of upstream signaling components and how intracellular pathways and physiology are regulated accordingly. In Part I, I present my work towards understanding upstream IIS pathway components using a systems biology approach. The goal of this study is to gain insight into the redundancy and specificity of the insulin gene family responsible for initiating IIS pathway activity in Caenorhabditis elegans. The information gained will serve as a foundation for future studies dissecting the molecular mechanisms of this pathway in efforts to uncouple the downstream signaling and physiological outputs. The clear impact of metabolism on aging and disease stimulated questions regarding the potential of promoting health and longevity through diet and dietary mimetics. Recent findings indicate reduced food intake, meal timing and nutritional modulation of the gut microbiome can ameliorate signs of aging and age-associated diseases. Aging, therefore, is also the result of dynamic and complex interplay between genes of an organism and its environment. In Part II, I will discuss my efforts to gain insight into how diet influences aging. This preliminary study has demonstrated that diet can affect lifespan in the model organism, C. elegans. Additionally, we observe diet-specific effects on drug efficacy that, in turn, modulates C. elegans lifespan and reproduction. The implications of these experiments, while limited, illustrate a potentially greater role in diet- and drug-mediated effects on lifespan.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20077号 / 医博第4170号 / 新制||医||1018(附属図書館) / 33193 / 京都大学大学院医学研究科医学専攻 / (主査)教授 江藤 浩之, 教授 斎藤 通紀, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM