Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 117-139).

Endothelial dysfunction is a pathologic state characterized by an altered equilibrium among vasodilatory and antithrombotic mediators and vasoconstrictive and prothrombotic mediators produced by the vascular endothelium. Multiple factors induce impaired production or increased consumption nitric oxide (NO), the key mediator of vascular homeostasis, produced by the nitric oxide synthase enzymes (NOS). Endothelial dysfunction represents one of the initial steps in the development of atherosclerosis, a chronic inflammatory disease of the vascular wall. The inducible enzyme heme oxygenase 1 (HO-1) represents one of the main cellular defense mechanisms against increased oxidative stress and decreased NO bioavailability accompanying endothelial dysfunction and atherosclerosis. We studied the genetic determinants of endothelial dysfunction and atherosclerosis by evaluating the association of the G894T endothelial NOS (eNOS) polymorphism and the HO-1 (GT)n promoter polymorphism with coronary artery atherosclerosis severity and risk profile and their evolution during hypolipidaemic treatment. In addition, we searched for genetic variations in exons 25 and 26 of eNOS gene, encoding the C-terminal part of the protein, deemed crucial for proper enzyme function and the 3'- untranslated region crucial for eNOS...

Apesar de sua importância clínica e epidemiológica, a fisiopatologia da préeclâmpsia ainda não foi completamente compreendida. Sabe-se que a doença constitui-se de uma fase pré-clínica e um estágio clínico. Durante a última década muito esforço tem se concentrado na identificação precoce da doença, ainda em sua fase pré-clínica. A literatura científica tem demonstrado claramente um desequilíbrio na regulação da angiogênese das gestantes com pré-eclâmpsia, marcado por níveis elevados do fator antiangiogênico soluble fms-like tyrosine kinase-1 (sFlt-1) e níveis diminuídos do fator pró-angiogênico placental growth fator (PlGF). Embora um número crescente de estudos em populações de alto risco tenha avaliado o papel desses biomarcadores no diagnóstico de pré-eclâmpsia, dados sobre sua utilização para a predição de pré-eclâmpsia superajuntada, cujo diagnóstico pode ser particularmente difícil, permanecem relativamente escassos e controversos. Com o presente estudo pretendemos avaliar o desempenho de medidas seriadas dos níveis maternos circulantes dos fatores sFlt-1 e PlGF, bem como da razão sFlt-1/PlGF, para predição de pré-eclâmpsia superajuntada e compará-lo ao seu desempenho na predição de pré-eclâmpsia em sua forma \"pura\", não superajuntada. Para este propósito, estudamos uma coorte prospectiva composta de dois braços, um de gestantes com hipertensão arterial crônica e outro de gestantes normotensas, e avaliamos os níveis séricos de sFlt-1 e de PlGF e a razão sFlt-1/PlGF nas idades gestacionais de 20, 26, 32 e 36 semanas, tendo como desfecho principal o diagnóstico de pré-eclâmpsia. Um total de 97 gestantes foram acompanhadas, 37 normotensas e 60 com hipertensão arterial crônica. Entre elas, 4 (10,8%) desenvolveram pré-eclâmpsia e 14 (23,3%) desenvolveram pré-eclâmpsia superajuntada. Para predição de pré-eclâmpsia, a análise ROC (Receiver Operating Characteristics) apresentou área sob a curva (AUC - area under curve) de 0,83 (IC 95% = 0,68-0,99, P = 0,035) para dosagem de PlGF com 20 semanas e AUC = 0,92 (IC 95% = 0,81 - 1,00, P = 0,007) para a razão sFlt-1/PlGF com 26 semanas de gestação. A variação percentual dos níveis de PlGF entre 26 e 32 semanas de gestação apresentou AUC = 0,96 (IC de 95% = 0,89-1,00, P = 0,003). Para a predição de pré-eclâmpsia superajuntada, a razão sFlt-1/PIGF na idade gestacional de 32 semanas apresentou AUC = 0,69 (IC de 95% = 0,53-0,85, P = 0,039). Entre 20 e 26 semanas de gestação, a variação percentual do PIGF e da razão sFlt-1/PlGF apresentaram, respectivamente, AUC = 0,74 (IC de 95% = 0,58-0,90, P = 0,018) e AUC = 0,71 (IC 95% = 0,52-0,91, P = 0,034). Por nossos resultados podemos concluir que, embora os níveis de PlGF e a razão sFlt-1/ PlGF tenham apresentado bons desempenhos na predição de pré-eclâmpsia, é preciso ter cuidado ao usá-los para a predição de pré-eclâmpsia superajuntada. Nessas gestantes, a dosagem dos fatores angiogênicos apresenta capacidade de predição menor e mais tardia. Avaliações seriadas dos fatores podem melhorar o desempenho dos testes para predição de pré-eclâmpsia superajuntada em idades gestacionais mais precoces / Despite being a major public health problem, the pathophysiology of preeclampsia is incompletely understood. Preeclampsia progression comprises a pre-clinical stage and a clinical stage. During the last decade much work has focused on identifying the pre-clinical stage of preeclampsia. Many researchers have clearly demonstrated an anti-angiogenic imbalance that is marked by higher levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and lower levels of placental growth factor (PlGF) in the subjects who develop preeclampsia compared with those who do not. Although a growing number of studies in the high-risk population have shown the role of these biomarkers in diagnosing preeclampsia, superimposed preeclampsia, which can be a challenging diagnosis, remains partially understudied and the literature regarding this subject continues to be relatively scarce as well as controversial. By this study, we aimed to evaluate the performance of serial measurements of maternal circulating sFlt-1 and PlGF levels for the prediction of superimposed preeclampsia in chronic hypertensive subjects and to compare it to the prediction of preeclampsia in normotensive control subjects. For this purpose, we evaluated a two-armed prospective cohort of women with normotensive and chronic hypertensive pregnancies and assessed the serum levels of sFlt-1 and PlGF and the sFlt-1/PlGF ratio at gestational ages of 20, 26, 32 and 36 weeks, having preeclampsia as the primary outcome to be predicted. A total of 97 women were followed-up, 37 in the normotensive group and 60 in the chronic hypertensive group. Among them, 4 (10.8%) women developed preeclampsia and 14 (23.3%) developed superimposed preeclampsia. For predicting preeclampsia, PlGF at 20 gestational weeks presented an AUC=0.83 (CI 95% = 0.68 - 0.99, P=0.035) and the sFlt-1/PlGF ratio at 26 gestational weeks presented an AUC=0.92 (CI95% = 0.81 - 1.00, P=0.007). The percent change of the PlGF levels between 26 and 32 gestational weeks presented an AUC=0.96 (CI 95% = 0.89 - 1.00, P=0.003). For predicting superimposed preeclampsia, the sFlt-1/PlGF ratio at 32 gestational weeks presented an AUC=0.69 (CI 95% = 0.53 - 0.85, P=0.039). Between 20 and 26 gestational weeks, the percent change of PlGF and the sFlt-1/PlGF ratio presented, respectively, an AUC=0.74 (CI 95% = 0.58 - 0.90, P=0.018) and an AUC=0.71 (CI 95% = 0.52 - 0.91, P=0.034). By our results, we concluded that, although the PlGF level and the sFlt-1/PlGF ratio present good performances in the prediction of preeclampsia, caution is required when using them for the prediction of superimposed preeclampsia. Sequential assessments slightly improve the test performances for predicting superimposed preeclampsia at earlier gestational ages

INTRODUÇÃO: O impacto do aumento de expressão do fator de crescimento endotelial no curso da Leucemia Mielóide Crônica (LMC) ainda é desconhecido, porém há relatos de que estes pacientes apresentam maior densidade vascular em medula óssea do que em indivíduos saudáveis, principalmente em crise blástica. Outro fator recentemente associado ao aumento da angiogênese é a expressão anormal da proteína hsecurina, que, por sua vez, inibi uma protease denominada separase, responsável pela separação das cromátides irmãs durante a anáfase da mitose. Por esses motivos, quantificamos células endoteliais circulantes e VEGF em portadores de LMC como marcador de angiogênese e expressão do gene hsecurina. MÉTODOS: Realizamos análise prospectiva e consecutiva de uma coorte de 31 pacientes com LMC em fase crônica ao diagnóstico, 23 em crise blástica, 30 em fase acelerada, atendidos no ambulatório de Hematologia da FMUSP e 50 indivíduos saudáveis, doadores de plaquetas por aférese, para quantificação da porcentagem de células endoteliais circulantes e subtipos pelo método de citometria de fluxo no laboratório de Imunopatologia HC/FMUSP. Desta coorte 25 pacientes em fase crônica, 14 em crise blástica, 26 em fase acelerada e 32 indivíduos saudáveis foram analisados para os genes hsecurina e VEGF por PCR quantitativo em tempo real. RESULTADOS: A mediana da porcentagem das células endoteliais circulantes foi de 0, 0146% em LMC em crise blástica e 0,0059% no grupo controle, p < 0,01 às custas das células endoteliais maduras (p < 0,01). A mediana de células endoteliais circulantes em crise blástica foi de 0, 0146%, superior à da fase acelerada (0,0059%), p < 0,01 com predomínio de células endoteliais maduras (p < 0,01). Em relação à expressão do gene VEGF observamos aumento estatisticamente significativo nas fases crônica (p < 0,01), acelerada (p < 0,01) e crise blástica (p = 0,04). Encontramos aumento significativo da expressão do gene hsecurina na crise blástica da doença, com mediana de 0,390 em relação aos grupos controle com mediana de 0,125 (p < 0,01) e fase acelerada, com mediana de 0,230 (p = 0,04). Os pacientes na fase crônica da doença apresentaram mediana de 0,260 e p = 0,03 quando comparados com o grupo controle. CONCLUSÃO: Observamos neste estudo que a quantificação de CEC é uma ferramenta útil para predizer e identificar precocemente a progressão da LMC para fase blástica, diferentemente da variável VEGF que foi elevado em todas as fases da doença. A expressão do gene hSecurina na fase crônica da doença foi significantemente alta, demonstrando provável relação com a elevação da taxa de proliferação celular. Entretanto, estudos complementares do gene hSecurina deverão ser realizados na crise blástica da LMC, para entendermos com precisão o real significado nesta fase da doença. / INTRODUCTION: The impact of the increased expression of vascular endothelial growth factor in the course of chronic myeloid leukemia (CML) is still unknown, but there are reports that those patients have higher vascular density in bone marrow than healthy individuals, particularly in blast crisis. Another factor recently associated with increased angiogenesis is the abnormal expression of protein hSecurin, which, in turn, inhibits a protease called separase, responsible for the separation of sister chromatids during the anaphase of mitosis. For these reasons, we quantified circulating endothelial cells and VEGF in patients with CML as a marker of angiogenesis and hSecurin gene expression. METHODS: We performed a prospective analysis of consecutive cases in a cohort of 31 patients with CML in chronic phase at diagnosis, 23 in blast crisis, 30 in accelerated phase who attended the outpatient Hematology FMUSP ward, and 50 healthy subjects, platelet apheresis donors, for quantification of the percentage of circulating endothelial cells and subtypes through the flow cytometry method, at HC/FMUSP Immunopathology laboratory. In this cohort, 25 patients in chronic phase, 14 in blast crisis, 26 in accelerated phase, and 32 healthy subjects were tested for the genes VEGF and hSecurin by quantitative real-time PCR. RESULTS: The median percentage of circulating endothelial cells was 0.0146% in CML in blast crisis and 0.0059% in the control group, p <0.01 at the expense of mature endothelial cells (p <0.01). The median circulating endothelial cells in blast crisis was 0.0146% higher than in accelerated phase (0.0059%), p <0.01 with predominance of mature endothelial cells (p <0.01). Regarding the expression of the VEGF gene, a statistically significant increase was observed in chronic phase (p <0.01), accelerated (p <0.01) and blast crisis (p = 0.04). We found a significant increase in hSecurin gene expression in blast crisis disease, with a median of 0.390 compared to control groups, with a median of 0.125 (p <0.01) and accelerated phase, with a median of 0.230 (p = 0.04). Patients with chronic disease had a median of 0.260 and p = 0.03 compared with the control group. CONCLUSION: In this study, we observed that the quantification of CPB is a useful tool to predict and identify the early progression of CML to blast phase, unlike the VEGF variable, which was elevated in all stages of the disease. The expression of hSecurin gene in chronic phase was significantly higher, demonstrating a likely relationship with the increased cell proliferation rate. However, further studies of hSecurin gene should be made in the blastic crisis of CML to understand precisely the real meaning at this stage of the disease.

A. Background<p><p>Skin wound healing (WH) is a dynamic and extremely determinate process of cellular, humoral and molecular mechanisms which begins directly after wounding and can last for years. WH is described as is an intricate process in which the skin (or another organ-tissue) repairs itself after injury. The process of skin WH occurs through the actions of an interplay of cells, growth factors and cytokines leading to wound closure.<p><p>WH occurs in three precisely and highly programmed phases: the inflammatory phase (day 0 to day 7) followed by the proliferative phase or vasculogenic phase (day 7 to day 21) and finally the remodeling phase (2 days - up to 2 years). For a successful healing, all three phases must occur in the proper sequence and time frame.<p><p>Many factors can interfere with one or more phases of the WH process, thus causing improper or impaired healing. The proliferation phase, in particular, requires the participation of various cells types such as fibroblasts, endothelial cells (ECs) and endothelial progenitor cells (EPCs), to produce a healthy well-vascularized granulation tissue for epithelization and wound closure.<p><p>A.1 Wound Healing And Obesity<p><p>In 2008, over 1.4 billion adults, 20 and older, were overweight. Of these, obesity has been shown to affect over 500 million people (OMS website). Moreover, the prevalence of obesity continues to rise, and by 2018, it is estimated that obesity will cost $ 347 billion annually.<p><p>Each year, in the US, approximately 33 million overweight and obese patients undergo surgery. Obesity causes a number of known health problems and increased post-surgical complications such as wound infection, dehiscence, hematoma and seroma. Surgeons anecdotally report WH complications among obese patients; however, little research has been conducted to investigate the mechanisms mediating impaired obesity-related WH. <p><p>Some previous work on diabetic patients and diabetic mice showed an imbalance between pro-oxydant and anti-oxydant genes as well as impaired EPCs proliferation and tube formation during the WH process. More then a hundred cytologic factors have been found to impair WH in the type 2 diabetic patient. It is a very complex and multifactorial problem involving decreased growth factors secretion, impaired keratinocyte and fibroblast functions, impaired EPs function, alteration of the macrophage function and granulation tissue synthesis, etc. <p><p>Based on these findings and because obesity is associated with the development of type 2 diabetes, we hypothetize that, impaired balance between pro-apoptotic/anti-apoptotic and pro- oxydant /anti-oxydant genes is involved in impaired WH. Furthermore, we hypothetize that impaired EPCs function leads to the perturbation of the proliferation phase of obesity impaired WH.<p><p>A.2. Wound Healing and Age<p><p>The world population is aging; by 2030, nearly 20% of Americans, (± 72 million people), will be 65 years old and older. In 2010, 17% of the European population was over the age of 65. By 2060, it is projected that the share of those aged 65 and over will rise to 30%, accounting for more then 150 million people. (ec.europa.eu) These aging subjects undergo an increasing number of surgical procedures: in the past two decades, the percentage of surgeries in patients over 65 has doubled to nearly 40%.<p>As a corollary, it is well established knowledge that elderly WH is impaired. However, little is known about the underlying mechanisms of age-related impaired WH.<p><p>As previously mentioned, adult BM-derived EPCs contribute to peripheral tissue repair and regeneration. In light of the abundant literature suggesting that neovascularization is impaired in the elderly, we characterize a novel model of senile cutaneous WH and investigate the role that vasculogenesis plays in the pathogenesis of age related impaired WH.<p>Aged mice colonies have traditionally been the model for aged small mammalian research, however, the ability to use a readily-available transgenic mouse model with features of accelerated aging would aid in the exploration of targeted therapies and a great number of age-related investigations.<p><p>We hypothesize that the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24-/-) mouse mimics physiological ageing and can be used as a novel model for the study of senescent WH. We further hypothetized that impaired balance between pro-apoptotic/anti-apoptotic and pro-oxydant /anti-oxydant genes as well as impaired EPCs function are responsible for the impairment of the proliferative phase, leading to overall impaired WH.<p><p>A.3 Aims<p><p>Recently, a great deal of research has been directed at understanding the critical factors inducing poorly healing wounds. However, a lot remains unclear.<p><p>It is now well accepted that new blood vessel formation occurs not only by angiogenesis (blood vessels formation from a preexisting network of capillaries), but also by vasculogenesis (blood vessels formation from BM SCs recruitment) and that EPCs contribute to as much as 25% of new blood vessels formed in healing tissues4. They are mobilized from the BM in response to injury and production of local cytokines, are incorporate into wounds and play an integral role in systemic tissue repair. <p><p>Based on this finding, we hypothesized that co-morbidities related impaired WH may be due, in part, to decreased EPCs number, migration/homing, and/or function resulting in impaired vasculogenesis. Because age and/or obesity have been shown to be one of the most common predictors of altered WH, we decided to focus on these two parameters.<p><p>Following a bedside to bench approach the purpose of this work was to 1) develop coherent and translatable models of co-morbidity digging in the physiologic/pathologic mechanisms underlying altered healing in obese and senile mice; 2) develop targeted therapeutics to improve impaired WH.<p><p>B. Material and Methods<p><p>B.1 Human Model<p><p>Since obesity impairs WH and BM EPCs are important for tissue repair, we hypothesize that obesity- impaired WH is due, in part, to impaired EPCs mobilization, trafficking, and function. Peripheral blood was obtained from non diabetic, obese (BMI > 30, n = 25), and non obese (BMI < 30, n = 17) subjects. Peripheral blood human EPCs were isolated, quantified, and functionally assessed.<p>As for aged impaired WH, EPCs of aged subjects have already been found to have decreased adhesion, migration and proliferative properties as well as being decreased in number in elderly patients undergoing surgery compared to younger patients.<p><p>B.2. Mice Models<p><p>Two models of WH were developed and characterized.<p>In order to isolate the effect of obesity on EPCs and WH, OB non-diabetic female TallyHo/JngJ mouse were selected (Female mice don’t express hyperglycemia and hyperinsulinemia). Female SWR/J non-OB mice were used as control mice. In order to limit variables, TallyHO/JngJ obese mice were selected over other OB mice that exhibit a polygenic type of obesity (Jackson Laboratory Website). By selecting this mouse model, we have excluded in our selection of the ideal model common confounding factors such as hyperglycemia, hyperinsulinemia, immune disorders.<p><p>Zmpste24 is a metalloproteinase involved in the maturation of lamin A (LmnA), an essential component of the nuclear envelope. When Zmpste24 or LmnA are knocked-out, mice exhibit profound nuclear architectural abnormalities and histopathological defects that phenocopy an accelerated aging process. Of crucial importance, the lamin-A dependent nuclear alterations seen in Zmpste24-deficient mice have also been found in human physiological aging. We defined the utilization of the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24- /-) mouse as a novel model for the study of senescent WH (controls used were C57BL/6J mice).<p><p>B.3. Wounding Model and Data Collection<p><p>All mice group underwent wounding using a stented wound model developed in our laboratory and previously published. Briefly, paired 6-mm circular, full-thickness wounds extending through the panniculus carnosus were made on the dorsal skin of the mouse. An O-ring, 12-mm splint made of silicone sheeting was then sutured to the skin around the wound. To minimize wound contraction and reliably recapitulated the granulation and re-epithelialization seen in human WH by secondary intention. Time to wound closure was measured using standardized digital photographs taken on days 0, 7, 14, and 21. Wound closure was calculated as a percentage of the original wound.<p><p>For each model, EPCs were harvested, quantified by flow-cytometry and their function tested. Wounds were harvested at various time points and RNA, DNA and protein analysis were conducted. Finally immunohistochemistry to assess epidermal thickness, vascularity and WH were also realized.<p><p>In a second step, after characterization of the models, local (using targeted siRNA gel) and systemic therapies (using AMD3100, a PC mobilizer) were applied on the wounds and compared to controls. WH was monitored. We conducted the previously mentioned analysis (RT-PCR, ELISA and DNA analysis) on the harvested samples.<p><p>All values are expressed as a mean ± standard error of mean (SEM). The number of mice per treatment group was determined using G*Power (G*Power©, Melbourne, Australia) to provide a power greater than 0.80. Student T test was realized to compare two groups among each other.<p><p>C. Results<p><p>C.1. Human EPCs Have Impaired Function<p><p>There was no difference in the number of baseline circulating human EPCs in non-diabetic OB and non-OB<p>subjects, but EPCs from OB subjects had impaired adhesion (p<0.05), migration (p<0.01), and proliferation (p<0.001).<p><p>C.2. Obesity and Wound Healing<p><p>TallyHo/JgnJ OB mice demonstrated significantly impaired healing when compared to SWR/J control mice. They healed at an average of 28 ± 2 days (p<0.05). Post-wounding circulating EPCs were quantified and wounds were analyzed. Circulating EPCs recruitment is impaired in wounded TallyHo/JngJ mice and their wounds shown significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Their wounds are characterized by increased apoptosis, increased DNA damage and impaired pro-/anti-oxydant balance. Immunonistochemistry and histology showed decreased vascular vessels in TallyHo/JngJ wounds and thinner epidermal thickness.<p><p>In the local treatment phase, local p53 silencing consistently improved WH to a nearly normal healing time (wounds healed in 18 ± 2 days, p<0.05). sip53 treatment showed a significant decrease in pro-apoptotic markers (p53, Bax, PUMA p<0.05) and a significant increase in angiogenic markers (VEGF, SDF-1α, HIF-1α) with increased blood vessel formation and decreased DNA damage.<p><p>C.3. Age and Wound Healing<p><p>In these experiments, we show that not only is Zmpste24-/- WH impaired when compared to C57BL/6J mice (Zmpste24-/- mice healed at average 40 days ± 2 days p<0.05) at all time points but that they also showed decreased vascularity and proliferation in the wound bed (p<0.05).<p><p>Histological analysis was performed utilizing hematoxylin and eosin staining to assess epidermal thickness, CD31 immunofluorescence to assess vascular density, p53 and caspase 3 to assess apoptosis, 8’OHdG staining to assess DNA damage and PCNA to assess proliferation. Epidermal thickness was significantly decreased in Zmpste24-/- animals compared to WT as well as vascular density, and proliferation in Zmpste24-/- wound tissue (p<0.05). <p><p>Circulating vasculogenic EPCs recruitment was impaired in Zmpste24-/- mice and their wounds showed significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Zmpste24-/- wounds are characterized by increased apoptosis and an abnormal rise in ROS.<p>In the treatment phase, local p53 silencing consistently improved healing by more then a two fold (18 ± 2 days). VEGF production was significantly increased and pro-apoptotic factors were significantly downregulated in siRNA-treated Zmpste24-/- mice (p<0.05). DNA damage due to ROS production was also shown to be significantly decreased following treatment. Our results suggest a vasculogenic dysfunction in wound closure and showed that the specific knock down of p53 significantly improves WH.<p><p>Because EPCs showed impaired function, lower peripheric blood counts and impaired SDF-1α/HIF-1α signaling, we hypothesized that improving their mobilization by using a progenitor cell mobilizer, AMD3100, known to mobilize SCs from the BM, in a systemic treatment phase will improve WH. Peripheral blood counts were significantly increased and time to wound closure significantly decreased (20 days ± 2, p<0.05). Vasculogenic markers and anti- apoptotic molecules were upregulated compare to non-treated animals.<p><p>D. Conclusions<p><p>Obesity impaired wound closure is a complex problem with many contributory factors. Our results suggest that obesity impairs the BM-derived EPCs response to peripheral injury and this, in turn, impairs wound closure. This impairment is associated with decreased new blood vessel formation and increased DNA damage leading to an increase in the p53 pathway. We also demonstrate that targeted siRNA therapy can partially rescue impaired WH due to obesity. Based on these results we support the encouraging argument that, WH and closure has the potential be improved through specific local and systemic therapies in vivo in our rodent model and that further studies are needed to support this in a clinical environment.<p><p>Impaired WH due to ageing is a complex phenomenon that is partially understood. We demonstrate that the Zmpste24-/- transgenic knockout mouse provides a model for age-related WH investigation. Zmpste24-/- animals heals their wounds with significant delays, showed impaired EPCs mobilization following wounding through an impaired HIF-1α/SDF-1α pathway and increased apoptosis. Furthermore, WH can be improved through specific local siRNA therapy and systemic stem cell mobilization therapies.<p><p>Our results suggest strong similar patterns between obesity and ageing in the way they mediate WH impairments trough (premature) ageing. Our encouraging endeavor to bring WH back to baseline in these diseased models underlines the possibility to reverse the microenvironment alterations and improves EPCs contribution to the WH process. Because EPCs are involved in virtually every tissue repair process happening in the human body, we hope that this work will lead the way for new research in various fields in medicine to improve wound care and quality of life of patients. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

L’endothélium joue un rôle primordial dans le développement et le maintien des multiples fonctions vasculaires. Il est ainsi largement impliqué dans des situations pathologiques comme les maladies cardio-vasculaires. La description de marqueurs endothéliaux circulants a permis une exploration non invasive de l'endothélium. Notre équipe s’est intéressée principalement aux cellules endothéliales circulantes (CEC), dont le taux reflète la lésion ou l’activation de l’endothélium, et aux progéniteurs endothéliaux circulants (PEC), marqueurs de régénération endothéliale. La découverte en 1997 par Asahara de la présence chez l’adulte de ces PEC, participant à la formation de nouveaux vaisseaux par vasculogenèse, a ouvert de nouvelles perspectives, notamment pour la thérapie cellulaire des pathologies ischémiques. Ce travail a consisté à développer les méthodes d’étude de ces cellules dans plusieurs contextes. Tout d’abord, nous avons exploré l’utilité de ces marqueurs dans la physiopathologie de l’hypertension artérielle pulmonaire (HTAP). Puis nous avons analysé le potentiel de mobilisation des progéniteurs endothéliaux à partir de la paroi vasculaire lors d’une ischémie locale chez des volontaires sains dans le cadre du développement d’un produit de thérapie cellulaire autologue. Une partie de ce projet a été de mettre en place et d’optimiser les techniques d’étude de ces marqueurs. Les CEC ont été quantifiées par immunoséparation magnétique (IMS), technique mise au point en 1992 (Dignat-George 1992) et transférée dans notre laboratoire. La quantification des PEC a été réalisée par cytométrie en flux et par culture cellulaire. En culture, deux types de PEC sont décrits : les PEC précoces, dont l’origine est monocytaire et pour lesquels la culture est déjà standardisée, et les « Endothelial Colony Forming Cells » (ECFC), seules cellules présentant des caractéristiques de cellules endothéliales progénitrices et pouvant être proposées comme produit de thérapie cellulaire. Nous avons optimisé la quantification des ECFC en culture en étudiant l’effet de diverses matrices et de la densité d’ensemencement des cellules mononucléées issues du sang total sur l’obtention de ces cellules et leurs propriétés angiogènes. La dysfonction endothéliale a été décrite comme un élément central dans le développement de l’HTAP dont le diagnostic repose sur la mesure de la pression artérielle pulmonaire par cathétérisme cardiaque droit. En l’absence de marqueur biologique non invasif dans cette maladie, nous avons quantifié les CEC et les progéniteurs circulants dans deux études. Une étude réalisée chez des patients adultes a montré une augmentation spécifique des CEC dans l’HTAP et non dans l’hypertension pulmonaire thromboembolique chronique. Ainsi les CEC semblent être le reflet des lésions endothéliales pulmonaires et non de la sévérité clinique des patients. L’autre étude a montré l’intérêt de la quantification des CEC dans la prise en charge thérapeutique des enfants souffrant d’HTAP secondaire à une cardiopathie congénitale, dont les formes irréversibles présentaient des taux élevés de CEC. Nous avons ainsi défini un nouveau marqueur non invasif à utilité diagnostique et pronostique. Les PEC sont des cellules rares dans le sang circulant, difficiles à expandre, et dont les essais de mobilisation médullaire se sont révélés insuffisants. L’hypothèse récente d’une réserve vasculaire des progéniteurs endothéliaux nous a conduits à étudier l’effet d’un processus d’ischémie locale sur la mobilisation de ces cellules chez des volontaires sains. Deux groupes d'âge ont été inclus afin d'évaluer l'impact du vieillissement sur la méthode de mobilisation étudiée. Malgré un effet de cette ischémie sur la dilatation endothéliale cette méthode n’a pas permis de mobiliser significativement les PEC issus de la paroi endothéliale, quel que soit l'âge des sujets. A l’inverse, l’hypoxie a eu un effet délétère sur les capacités angiogènes des ECFC. / The endothelium plays a key role in the development and the homeostasis of vascular functions. It is also well involved in pathological situations like cardiovascular diseases. Thanks to the description of circulating endothelial markers, non invasive study of the endothelium is now possible. Our group was particularly interested in circulating endothelial cells (CECs), the level of which reflects an endothelial activation or lesion, and to circulating endothelial progenitors cells (EPCs), markers of endothelial repair. EPC description by Asahara in 1997 in adult blood, involved in new blood vessel formation by vasculogenesis, offered new perspectives, specially for cell therapy in ischemic diseases. This work consisted in the development of methods to study these markers in different contexts. First, we explored the interest of these markers in the physiopathology of pulmonary arterial hypertension (PAH). Then we evaluated endothelial progenitors mobilization from the vascular wall by a local ischemia process in healthy volunteers, in the perspective of an autologous cell therapy product development. One part of this project was the implementation and optimization of the methods to study CEC and EPC. CEC were quantified by magnetic immunoseparation. This technique was developped in 1992 by F. Dignat-George's group and transferred in our laboratory. EPC were quantified by flow cytometry and cell culture. Two types of EPC are described in culture: the early EPC, which originate from monocyte lineage and which culture is standardized, and the « Endothelial Colony Forming Cells » (ECFC), the only cells presenting endothelial progenitor cell properties and which use as a cell therapy product can be considered. ECFC quantification by culture was optimized by assessment of the impact of diverse matrices and seeding concentrations of mononuclear cells isolated from whole blood, on ECFC commitment and their angiogenic properties. Endothelial dysfunction was described as a central element in the development of PAH, which diagnosis is based on the use of right heart catheterization. Due to the lack of noninvasive marker for this disease, CEC and circulating progenitors were quantified in two studies. One of them realized in adult patients showed a specific increase of CEC in PAH and not in post-embolic PH. CEC would then reflect the presence of specific endothelial lesions and not the clinical state of the patients. The other study demonstrated the interest of CEC quantification in the therapeutic care of children with PAH secondary to congenital heart disease, for whom patients in irreversible state had a higher level of CEC. We then defined a new noninvasive biomarker.that can be used for the diagnosis and prognosis of PAH. EPC are rare events in whole blood, difficult to expand and for which, mobilization protocols revealed insufficient. The recent hypothesis of a vascular reservoir for endothelial progenitor led us to study the effect of a local ischemia procedure on the mobilization of these cells in healthy volunteers. Two age groups were included to assess the impact of aging on this procedure. Despite a significant endothelial dilation with the local ischemia, no EPC were mobilized, whatever the age group. Ischemia even altered ECFC angiogenic properties.

Angiogenesis, the process of new vessel formation, appears to be a central mechanism that underlies the development of haemangiomas. Recently, intralesional bleomycin injection was used to treat paediatric haemangiomas with very good results. The purpose of this study was to determine whether there was significant systemic circulatory spill-over of bleomycin in haemangioma patients treated with intralesional bleomycin to determine safety of use. Furthermore, in order to elucidate bleomycin’s mechanism of action in inducing haemangioma regression, this study aimed at determining the effects of bleomycin on aspects of angiogenesis, namely, endothelial cell migration, growth and apoptosis, and comparing these effects with those of drugs previously reported to inhibit various aspects of the angiogenic process (mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D). Lastly, the effects of bleomycin, mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D were studied in an animal haemangioma model. A rapid and highly sensitive high performance liquid chromatographic (HPLC) method was developed. Blood samples were collected from four haemangioma patients before and after (over a 24 hour period) intralesional bleomycin (IB) therapy. As a control, blood samples were also collected at identical time intervals from four patients undergoing intravenous (IV) bleomycin chemotherapy for various malignant tumours. The HPLC method was used to quantitate bleomycin fractions in patient samples. The mean bleomycin concentration detected in plasma samples obtained from IB treated patients was 0.00 ìg/ml for both bleomycin A<Sub>2 and B2 over the 24-hour period following therapy. Plasma bleomycin A2 and B2 levels of 360.79 and 158.85 ìg/ml respectively were detected in samples obtained from cancer patients treated with bleomycin IV. These findings indicate that the low levels detected may translate to a significantly lesser risk of pulmonary fibrosis following IBI. The effect of drugs on endothelial cell migration was analyzed by wounding a confluent monolayer of cells and determining the number of cells that had migrated from the wound edge. Endothelial cell growth was determined in cells treated with various drug concentrations while apoptosis was examined using hematoxylin and eosin staining, DNA fragmentation assay and acridine orange staining. The effect of test drugs on in vitro angiogenesis was determined on endothelial cells induced to form capillary-like tubes in collagen gel. Test drugs were then evaluated for antitumour activity in an animal haemangioma model. Data demonstrated that test drugs inhibited endothelial cell migration, with the exception of mitomycin C. All test drugs induced a reduction in the percentage of viable endothelial cell in a dose-dependant manner, and also induced endothelial cell apoptosis. The drugs inhibited angiogenesis in vitro and inhibited tumour development in vivo with varying potency. In general, results from this study indicated that there was negligible systemic spill-over of bleomycin following IB administration in patients with haemangiomas, suggesting a much lesser risk of developing bleomycin-induced pulmonary fibrosis. This study also showed that test drugs inhibited angiogenesis in vitro and haemangioma development in vivo in a mouse model. Taken together, these observations demonstrate that bleomycin may inhibit haemangioma growth by inhibiting angiogenesis. In addition, mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D may have potential in the treatment of haemangiomas of infancy, and should be investigated further in a murine haemangioma model to determine effective dose schedules. / Thesis (PhD)--University of Pretoria, 2009. / Physiology / unrestricted

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