Peter Carmeliet

Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis, and thrombolysis.

The effects of plasminogen activator inhibitor-1 (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-1-deficient (PAI-1-/-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1-/- and from PAI-1 wild type (PAI-1+/+) mice underwent limited but significantly different (P < 0.001) spontaneous lysis within 3 h (6 +/- 1 vs 3 +/- 1%, respectively). A 25-microliters 125I-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47 +/- 5, 66 +/- 3, and 87 +/- 7% within 8 h in PAI-1+/+, heterozygous PAI-1-deficient (PAI-1+/-), and PAI-1-/- mice, respectively (P = 0.002 for PAI-1+/+ vs PAI-1-/- mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PAI-1+/+ and PAI-1-/- mice, were 35 +/- 5 and 91 +/- 3% within 4 h, respectively (P < 0.001). 11 out of 26 PAI-1+/+ but only 1 out of 25 PAI-1-/- mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 micrograms endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PAI-1-/- mice after partial amputation of the tail or of the caecum. Thus, disruption of the PAI-1 gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis.

Plasminogen activator inhibitor-1 gene-deficient mice. I. Generation by homologous recombination and characterization.

Homozygous plasminogen activator inhibitor-1 (PAI-1)-deficient (PAI-1-/-) mice were generated by homologous recombination in D3 embryonic stem cells. Deletion of the genomic sequences encompassing the transcription initiation site and the entire coding regions of murine PAI-1 was demonstrated by Southern blot analysis. A 3.0-kb PAI-1-specific mRNA was identified by Northern blot analysis in liver from PAI-1 wild type (PAI-1+/+) but not from PAI-1-/- mice. Plasma PAI-1 levels, measured 2-4 h after endotoxin (2.0 mg/kg) injection were 63 +/- 2 ng/ml, 30 +/- 10 ng/ml, and undetectable (< 2 ng/ml) in PAI-1+/+, heterozygous (PAI-1+/-) and PAI-1-/- mice, respectively (mean +/- SEM, n = 4-11). PAI-1-specific immunoreactivity was demonstrable in kidneys of PAI-1+/+ but not of PAI-1-/- mice. SDS-gel electrophoresis of plasma incubated with 125I-labeled recombinant human tissue-type plasminogen activator revealed an approximately 115,000-M(r) component with plasma from endotoxin-stimulated (0.5 mg/kg) PAI-1+/+ but not from PAI-1-/- mice, which could be precipitated with a polyclonal anti-PAI-1 antiserum. PAI-1-/- mice were viable, produced similar sizes of litters as PAI-1+/+ mice, and showed no apparent macroscopic or microscopic histological abnormalities.

Biological Effects of Disruption of the Tissue-Type Plasminogen Activator, Urokinase-Type Plasminogen Activator, and Plasminogen Activator Inhibitor-1 Genes in Micea

Mammalian blood contains an enzymatic system, called the fibrinolytic system or the plasminogen/plasmin system, that has been claimed to play a role in several phenomena associated with proteolysis, including blood clot dissolution (thrombolysis), thrombosis, hemostasis, atherosclerosis, ovulation, embryo implantation, embryogenesis, wound healing, malignancy, and brain function.M The fibrinolytic system comprises an inactive proenzyme, plasminogen, which is activated to the proteolytic enzyme plasmin by two physiological plasminogen activators, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Inhibition of the fibrinolytic system may occur at the level of plasmin, mainly by a2-antiplasmin, or at the level of the plasminogen activators by specific plasminogen activator inhibitors (PAIs). Of those, endothelial or type-1 PAI-1 (PAI-1) appears to be the primary physiological inhibitor of plasminogen activation.’ t-PA is believed to be primarily responsible for removal of fibrin from the vascular tree; it has a specific affinity for fibrin, and produces clot-restricted plasminogen activation. The role of u-PA in thrombolysis is less well defined; it lacks affinity for fibrin and requires conversion from a single-chain precursor to a catalytically active two-chain der i~a t ive .~ Whether other plasminogen activation pathways, such

Retinal Angiogenesis and Growth Factors

The vasculature is the first organ to arise during development. Blood vessels run through virtually every organ in the body, ensuring metabolic homeostasis by supplying oxygen and nutrients and removing waste products. Consequently, a dysfunction of blood vessels compromises normal organ performance. This in turn may lead to congenital or acquired diseases, disability or even death. The lymphatic system develops in parallel but secondary to the blood vascular system. It serves an essential function in absorbing and transporting tissue fluid and extravasated proteins and cells back to the venous circulation. Understanding the principles of how blood and lymph vessels form and which angiogenic factors are involved might provide novel attractive opportunities for treatment of angiogenic disorders.

Molecular and Cellular Angiogenesis

Blood vessels deliver oxygen and nutrients — essential for survival of cells in multicellular organisms. Since the diffusion for oxygen is limited, cells in the mammalian body are located within a short range of blood vessels. The growth of new blood vessels via vasculogenesis or angiogenesis is tightly regulated by an intricate balance between activators and inhibitors. In a normal adult, angiogenesis cyclically occurs during reproduction and as part of a self-limiting reparative program after wounding or during inflammation. The remainder of the adult vasculature is quiescent, with only 0.01% of endothelial cells undergoing cell division. During pathological conditions, angiogenesis occurs in an uncontrolled manner. Leonardo Da Vinci was among the first to demonstrate angiogenesis in an inflamed human lung. We now know that abnormal vascular growth or function also contributes to numerous nonneoplastic disorders of significant morbidity, and the list is expanding every day (Table 1). For example, an excess of vessels in diabetic retinopathy can lead to blindness or promote cancer.

Molecular Analysis of Vascular Development and Disorders

Blood vessels are among the first organs to develop during embryogenesis and are essential for organogenesis and nutrition of the embryo. Although formation of blood vessels most actively occurs during embryonic development, tissue vascularization proceeds after birth in the retina, in the heart, and, cyclically, in the reproductive organs. In addition, abnormal vessel growth importantly contributes to the pathogenesis of several disorders with high morbidity and mortalitiy. Excessive vessel growth has been implicated in the pathogenesis of retinopathies, cancer and inflammation. In contrast, insufficient vessel growth may lead to tissue ischemia and failure. Although formation of new blood vessels has typically been associated with endothelial cells, the periendothelial mural pericytes (smaller vessels) or smooth muscle cells (larger vessels) are equally important for normal and pathological vessel formation. Recent studies have highlighted the importance of endothelial periendothelial cell crosstalk during normal and pathological blood vessel formation. Accordingly, integrated research into the molecular mechanisms that regulate the development and function of both endothelial and pericyte/smooth muscle cells has become a major focus in vascular biology. A number of candidate molecules (growth factors, matrix component, adhesion molecules, and proteinases) has been identified that stimulate or inhibit these processes. Recently, remarkable progress in their molecular analysis has been achieved through targeted manipulation of the mouse genome. The role of some of these molecules will be discussed in this chapter.

328 Role of the Fragile X Mental Retardation Protein in Tumor Progression and Metastasis Formation

pression in isogenic normal (MRC5), immortal (pre-malignant) (MRC5hTERT) and tumorigenic (MRC5hTERT TZ) human myofibroblasts, which are p53defective, as well as tumour-derived fibrosarcoma cells (HT1080). Results and Discussion: SiRNA-mediated repression of DKC1 or TERT expression induced acute proliferative arrest of immortal and tumorigenic cells, while having no adverse effect on the proliferation of isogenic normal cells. Furthermore, suppression of DKC1 or TERT gene expression also impaired anchorage-independent growth of tumorigenic cells. In comparison, treatment with a control siRNA, siRNA targeting TERC or the small molecule telomerase enzyme inhibitor BIBR1532, had no acute effect on proliferation in these short term assays. While the growth arrest mediated by DKC1 or TERT repression was not dependent on p53 function, loss of p53 function altered the cell cycle phase in which the cells arrested. The growth arrest induced by DKC1 repression remained unaffected by TERC overexpression suggesting a telomere independent role for DKC1. Microarray analysis of gene expression and Gene Set Enrichment analysis (GSEA) of siRNA-transfected normal, immortal and tumorigenic MRC5 cells was employed to gain insight to the mechanisms that underpinned the acute anti-proliferative effects of TERT and DKC1 repression. Investigations are currently underway to determine the effect of stable repression of TERT and DKC1 on subcutaneous tumour formation in immunocompromised mice. Conclusions: Together, these results demonstrate that telomeraseimmortalised pre-malignant and tumorigenic cells required continued expression of TERT and DKC1 for replication. This provides further evidence of telomere length-independent functions of TERT and DKC1 in these cells since there was insufficient time for telomere shortening to occur. The potent and specific anti-proliferative effects resulting from repression of DKC1 and TERT in immortal and tumorigenic cells demonstrate that directly targeting these telomerase components has potential as a potent and specific approach to treatment of the broad spectrum of cancers that express telomerase.

CHAPTER 19 – VEGF, an Angiogenic Factor with Neurotrophic Activity, Useful for Treatment of ALS?

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. Recent evidence indicated, however, that VEGF also plays a significant role in the development and maintenance of the neural system. In addition to its ability to promote the survival of various populations of neurons and glial cells, transgenic mice expressing low levels of VEGF develop late-onset motoneuron degeneration, reminiscent of amyotrophic lateral sclerosis (ALS) in humans. Recent data further revealed that intracerebroventricular (ICV) delivery of recombinant VEGF protein delays disease onset, improves motor performance and prolongs survival of ALS rats. Notably, intramuscular administration of a VEGF-expressing rabies G-pseudotyped lentivirus, which is retrogradely transported to the neuronal cell body, increased the life expectancy of ALS mice by as much as 30%. Together with the positive effects achieved with viral gene delivery for insulin-like growth factor 1 (IGF1), these efforts have primed widespread interest in applying these vectors for therapeutic use in ALS patients.

Mouse Models to Study Pro-and Antiangiogenic Potential: Novel Roles for PLGF and FLT1

Angiogenesis, or the formation of new blood vessels, is a complex, yet highly regulated process that is crucial for normal development and physiology, but when disturbed, contributes to the pathogenesis of at least 70 clinical disorders16,31,51. Millions of patients would therefore benefit from therapeutic modulation of blood vessel growth, if effective and specific stimulators and inhibitors could be identified. In spite of the fact that over 40 pro-angiogenic and anti-angiogenic compounds have been evaluated in clinical trials, none has yet been approved for general use.

42 – Formation of Blood and Lymphatic Vessels: Role of Progenitors

This chapter demonstrates how functional blood and lymphatic vessels form in the embryo and the adult from vascular stem/progenitor cells, and how this knowledge has contributed to the emergence of several stem/progenitor cell-based approaches to stimulate vessel growth in preclinical and clinical settings. Nearly all tissues in higher vertebrates are supplied by and dependent on two largely separated but functionally complementary vascular circuits, one carrying blood cells for oxygen and nutrient delivery and the other recirculating immune cells and extravasated tissue fluid or lymph. Not surprisingly, developmental defects in either of these important systems are usually incompatible with life or cause severe morbidity. In addition, the dysfunction or lack of blood or lymphatic vessels in later life underlies numerous life-threatening diseases, such as ischemic disorders and lymphedema. Therefore, unraveling the cellular and molecular players in the formation of both vascular systems is invaluable for designing therapeutic interventions to correct blood or lymphatic vessel defects.