No description available.

No description available.

Angiogenese ist entscheidend für die meisten physiologische Prozesse und viele pathologische Umstände. Dabei wird Angiogenese durch die Interaktion zwischen der extrazellulären Matrix (ECM) und endothelialen Zellen reguliert. Während der kardiovaskulären Entwicklung im Zebrafisch fördert Klf2, ein blutstrom-sensitiver Transkriptionsfaktor, die VEGF-abhängige Angiogenese. Der Mechanismus, bei dem biophysikalische Reize die Klf2 Expression regulieren und Angiogenese kontrollieren, ist größtenteils unbekannt. In meiner Studie zeige ich, dass erhöhte klf2 mRNA Expression den molekularen und morphogenetischen kardiovaskulären Defekten in Zebrafisch ccm Mutanten zugrundeliegen. Desweiteren zeige ich, dass diese Defekte durch verstärkte egfl7-Expression und Angiogenese vermittelt werden. Meine Studie zeigt ausserdem, dass die Klf2-Expression unabhängig vom Blutstrom durch den Extrazellularmatrix-bindenden Rezeptor beta1 Integrin reguliert wird. Der CCM-Protein-Komplex, zusammen mit dem ihm verbundenden Integrin-regulierenden Protein ICAP-1 verhindert ein verstärktes Angiogenese-Signal in endothelialen Zellen, indem es die beta1 Integrin-abhängige Klf2 Expression begrenzt. Zusammenfassend zeigt meine Arbeit einen neuen beta1 Integrin-Klf2-Egfl7 Signalweg, der durch zerebrale kavernöse malformations (CCM) Proteine reguliert wird / Angiogenesis is critical to most physiological processes and many pathological conditions. This process is controlled by physical interactions between the extracellular matrix (ECM) and endothelial cells. Klf2, a blood flow–sensitive transcription factor, promotes VEGF-dependent angiogenesis during zebrafish cardiovascular development. However, the mechanism by which biophysical stimuli regulate Klf2 expression and control angiogenesis remains largely unknown. In my study, I show that elevated klf2 mRNA levels underlie the molecular and morphogenetic cardiovascular defects in zebrafish ccm mutants. Furthermore, I demonstrate that these defects are mediated by enhanced egfl7 expression and angiogenesis signaling. My study also revealed that Klf2 expression is regulated by the extracellular matrix-binding receptor beta1 integrin in the absence of blood flow. The CCM protein complex and its associated beta1 integrin-regulatory protein ICAP-1 prevents increased angiogenesis signaling in endothelial cells by limiting beta1 integrin-mediated expression of Klf2. ln sum, my work uncovered a novel beta1 integrin-Klf2-Egfl7 signaling pathway, which is regulated by the cerebral cavernous malformations (CCM) proteins.

Krüppel-like factors (KLFs) are a family of 3 Cys2/His2 zinc finger transcription factors with a diverse set of roles in cellular differentiation, cell cycle regulation, tumor suppression, erythropoiesis, angiogenesis, and other processes. During embryonic development, KLF2 has a role in vessel maturation. Adult conditional KLF4 knockout mouse embryos have thickened arterial intima follow vascular injury. Breeding KLF2+/- and KLF4+/- mice resulted in the generation of KLF2/KLF4 double knockout (DKO) embryos. KLF2/KLF4 DKO embryos died by E10.5 with cranial bleeding. Using immunohistochemistry, embryo whole-mounts were examined for differences in gross vascularization between wild-type (WT), KLF2-/- and KLF2/KLF4 (DKO embryonic day 9.5 (E9.5) embryos. No obvious gross capillary abnormalities were noted in E9.5 KLF2/KLF4 DKOs, although the posterior cardinal vein appeared to narrow rostral to caudal in KLF2-/- and KLF2/KLF4 DKO embryos. Light and electronic microscopy were employed to investigate potential structural and ultrastructural phenotypes in KLF2/KLF4 DKO embryos. Microscopy confirmed hemorrhaging near and endothelial breaks in the primary head vein (PHV) in E9.5 KLF2/KLF4 DKOs (n=8) and E10.5 KLF2-/-KLF4+/- embryos (n=1). Electron micrographs illustrated a disrupted endothelium in KLF2/KLF4 DKOs with endothelial cells having filopodia-like projections. Surprisingly, KLF2-/- embryos had the presence of wider medial PHV endothelial gaps compared to WT at the electron micrograph level. Density counts revealed a 15% reduction in midline cranial mesenchyme at the level of hemorrhaging in KLF2/KLF4 DKOs compared to KLF2-/- (n=3). An in-situ hybridization localized KLF2 RNA expression to the endothelium of the PHV. A quantitative reverse transcriptase polymerase chain reaction assay revealed that the eNOS expression is synergistically regulated by KLF2 and KLF4, as a shared downstream target. It is proposed that KLF2 and KLF4 share in the regulation of multiple gene targets, leading to early death by E10.5.

Krüppel-like factors KLF1 and KLF2 are closely related transcription factors with three zinc finger domains in their carboxy-termini. KLF1 (erythroid Krüppel-like factor, or EKLF) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β- globin genes. KLF1 and KLF2 have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double knockout (KO) mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, and double KO embryos are more anemic and die sooner than either single KO. We have shown that KLF1 and KLF2 positively regulate the human ϵ- (embryonic) and γ-globin (fetal) genes during embryonic erythropoiesis. Chromatin immunoprecipitation assays (ChIP) show that KLF1 and KLF2 bind to the promoters of the human ϵ- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region (LCR) in mouse embryonic erythroid cells. ChIP assays show that KLF1 but not KLF2 ablation results in abnormal histone modifications in the β-globin locus in mouse embryonic erythroid cells. H3K9Ac and H3K4me3, which correlate with open chromatin and active transcription, are both reduced in KLF1-/- primitive erythroid cells. Human CD34+ hematopoietic stem cells obtained from umbilical cord blood were in vitro differentiated along the erythroid lineage. ChIP assays indicate that both KLF1 and KLF2 bind to the promoter of γ-globin gene in this fetal erythroid model. KLF1 knockdown in these cells affects mainly adult β- globin gene expression. However, the decrease in β- globin gene expression in KLF1 knockdown also affects the ratio of γ- to β- globin in these cells. H3K9Ac and H3K4me3 were decreased only at the β- globin gene which coincides with lower recruitment of RNA polymerase II and its active form, RNA polymerase II phospho-serine 2. In conclusion, we showed using mouse primitive erythroid cells and cord blood definitive cells that KLF1 and KLF2 coordinate the regulation of the mouse and human β- globin genes by direct binding to the promoters and LCR in the β- globin locus. In conclusion, cord blood hematopoietic cells could serve as a complimentary system in addition to the transgenic mouse models to study the regulation of γ- globin gene expression.

Hemoglobinopathies are some of the most common monogenic disorders in the world, affecting millions of people and representing a growing burden on health systems worldwide. Although the pathophysiology of sickle cell anemia and beta-thalassemia, two of the most common hemoglobinopathies, have been the focus of much research over the last century, patients affected by these diseases still lack a widely applicable and easily available cure. Sickle cell anemia and beta-thalassemia are caused by defects in the structure and production of the beta-globin chains that, along with the alpha-globin chains make up the heterotetrameric hemoglobin molecule. Studies geared towards re-expression of the silenced fetal gamma-globin gene in adult erythroid cells as a therapeutic strategy to alleviate the symptoms of beta-globin deficiencies have met with some success for the treatment of sickle cell anemia but not for beta-thalassemia. A better understanding of normal gamma-globin gene regulation will undoubtedly advance the development of more effective therapeutic strategies. Because many of the potential targets that may be modulated to achieve gamma-globin re-expression also have functions in erythroid cells other than regulating the gamma-globin gene, it is imperative to understand their role in all aspects of erythropoiesis before they are used for therapy. The current study focuses on the role of two Krüppel-like transcription factors, KLF1 and KLF2, which have known roles in the processes of primitive and definitive erythropoiesis as well as globin gene regulation. The regulation of primitive erythropoiesis by KLF1 and KLF2 is studied using the mouse as a model system because it is not possible to study primitive erythropoiesis in humans. Previous studies have shown that KLF1 and KLF2 are essential for and have overlapping roles in primitive erythropoiesis. Simultaneous ablation of KLF1 and KLF2 results in a severely anemic embryonic phenotype that is not evident in KLF1 or KLF2 single knockout embryos. In this study, we show that this anemia is caused by a paucity of blood cells, and exacerbated by diminished beta-like globin gene expression. The anemia phenotype is dose-dependent, and interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining both normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. It was discovered that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation, through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous deficiency of KLF1 and KLF2, than with loss of a single factor. The regulation of human beta-like globin gene expression is studied using a recently developed in vitro system for the production of erythroid cells from umbilical cord blood hematopoietic precursor cells, representing a more “fetal” model of globin gene expression. Previous studies have shown that KLF1 binds to the promoters of the gamma- and beta-globin genes, while KLF2 binds to the promoter of the gamma-globin gene in cord blood-derived erythroid cells. Studies using transgenic mice carrying the entire human beta-globin locus had indicated that KLF1 and KLF2 positively regulate gamma-globin expression in mouse embryonic erythroid cells. We demonstrate in this study that KLF1 appears to have dual roles in the regulation of gamma-globin expression in human cord blood-derived definitive erythroid cells. Partial depletion of KLF1 causes elevated gamma-globin expression, while nearly complete depletion of KLF1 results in a down-regulation of gamma-globin expression. Of particular interest was the observation that KLF2 positively regulates gamma-globin expression in cord blood-derived erythroid cells. Surprisingly, KLF2 also positively regulates beta-globin expression in these cells. If regulation of gamma-globin by KLF2 proves to be a direct effect, KLF2 will join a very small group of factors known to directly activate gamma-globin expression.

Krüppel like factors (KLFs) are a family of 17 proteins whose main function is gene regulation by binding to DNA elements in the promoters of various genes. KLF transcription factors recognize CACCC-elements and act as activators or repressors of the gene expression. Among the 17 family members, KLF1, KLF2, and KLF4 share high homology to each other. KLF1 is the founding member of the family and is an erythroid-specific protein. KLF2 is expressed in erythroid, endothelial, and other cells. KLF4 is expressed in endothelial, smooth muscle, and other cells. In this thesis, the functions of these KLFs were reviewed in the context of subjects related to erythropoiesis and cardiovascular development. A mouse model lacking KLF1, KLF2, and KLF4 was used to investigate whether these genes have overlapping functions in regulating the embryonic β-globin genes during early embryogenesis. Quantitative RT-PCR assays were used to measure the expression level of Ey- and βh1- globin mRNA at embryonic day 9.5 (E9.5). It was found that KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos express significantly decreased amounts of Ey- and βh1-globin genes when compared to WT and KLF4-/- embryos. There were no significant changes in the levels of Ey- and βh1-globin mRNA between KLF1-/-KLF2-/- and KLF1-/-KLF2-/-KLF4-/- embryos. It was demonstrated here that KLF1 does not regulate KLF2 in mouse erythroid cells at E10.5.

No description available.

Krüppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress, such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death by mouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental role for KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF2-/- embryos die earlier, by E11.5. E9.5 FVB/N KLF2-/- hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normal single layer. By E10.5, traditional and endothelial-specific FVB/N KLF2-/- AV cushions are hypocellular, suggesting that the cells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF2-/- hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number of mesenchymal cells migrating from FVB/N KLF2-/- AV explants into a collagen matrix is reduced considerably compared to wild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/N KLF2-/- embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF2-/- hearts have largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF2-/- embryos have a delay in the formation of the atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucose dehydrogenase (UGDH), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and UGDH promoters in chromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. Thus KLF2 plays a role in EMT, through its regulation of important cardiovascular genes. E10.5 FVB/N KLF2-/- embryos show gaps in the endothelial lining at the dorsal aorta and a number of blood cells localized outside the aorta suggesting either hemorrhaging or inability of the hematopoietic progenitors to reach the aortic endothelium and enter circulation. This is not observed in KLF2-/- embryos in a mixed genetic background. In conclusion, KLF2-/- cardiovascular phenotypes are genetic background-dependent. KLF4 is another member of the Krüppel-like transcription factor family phylogenetically close to KLF2. It is known to play an important role in vascular regulation. Our studies show that in vascular development KLF4 plays a complementary role to KLF2, indicated by cranial hemorrhaging in E9.5 KLF2-/-KLF4-/- embryos in an undefined mixed background. This phenotype is absent in either of the single knockouts. The role of KLF2 and KLF4 in vascular development has not been studied as much as adult vascular regulation. This study begins to define the roles of these two transcription factors in development of blood vessels. Congenital heart and valve defects are a common cause of infant mortality. KLF2 has never been studied in this context. Thus this work is important for a better understanding of the biology of valve development.

El endotelio disfuncional presenta, entre otras caracteristicas, alteración en los mecanismos de vasodilatación, complicaciones trombóticas, disminución de la resistencia al estrés oxidativo, aumento de la expresión de moléculas de adhesión y de la secreción de moléculas proinflamatorias. El factor de transcripción endotelial KLF2 juega un importante papel en la regulación del fenotipo protector endotelial y su expresión depende de las fuerza hemodinámicas generadas por el flujo sanguíneo y de la administración exógena de estatinas. La hipertensión portal y el daño hepático por I/R son dos condiciones patológicas asociadas a disfunción endotelial. Los trastornos estructurales característicos de la cirrosis hepática, la mayor causa prevalente de hipertensión portal en nuestro entorno, se acompañan de variaciones en las fuerzas hemodinámicas que pueden modificar la expresión de KLF2 y su programa transcripcional vasoprotector. Asímismo, durante la isquemia asociada a la preservación de injertos hepáticos para transplante, la interrupción de las fuerzas hemodinámicas generadas por el flujo sanguíneo podría resultar en la reducción de los programas endoteliales vasoprotectores, que se debería en parte a la pérdida de expresión de KLF2. Los trabajos de investigación de la presente tesis doctoral amplian el conocimiento de los mecanismos moleculares responsables de la disfunción endotelial hepática, demostrando: 1. Que KLF2 está muy expresado en los hígados cirróticos y que su expresión se induce en las fases tempranas de la progresión de la enfermedad, representando un mecanismo compensador para mejorar los desórdenes vasculares característicos de los hígados cirróticos. 2. Que los hígados preservados en condiciones de transplante muestran un descenso tiempo-dependiente de KLF2, acompañado de daño hepático y aumentada resistencia vascular. Además, demostran que la modulación farmacologica de la expresión de KLF2 puede ser beneficiosa tanto en el tratamiento de la hipertensión portal como en la preservación de los injertos hepáticos para transplante.