Search

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid β-oxidation, impairs lymphatic development. LECs use fatty acid β-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid β-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1–p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.

In our graying world population, we are increasingly facing brain injuries and age-associated neurodegenerative diseases, which are often characterized by axonal pathology. Here, we propose the killifish visual/retinotectal system as a model for investigating central nervous system repair, more specifically axonal regeneration, in an aging context. We first describe an optic nerve crush (ONC) injury paradigm in killifish to induce and study both de- and regeneration of retinal ganglion cells (RGCs) and their axons. Subsequently, we summarize several methods for mapping different steps of the regenerative process—namely, axonal regrowth and synapse reformation—using retro- and anterograde tracing methods, (immuno)histochemistry, and morphometrical analyses.

Loss of vision is a prominent feature of aging and vision is considered by many to be the most valuable sense to be lost. In our graying society, we are increasingly challenged by age-related deterioration of the central nervous system (CNS), as well as by age-associated neurodegenerative diseases and brain injuries, all often affecting the visual system and thus its performance. Here, we describe two visually driven behavior assays to evaluate visual performance upon aging or CNS damage in the fast-aging killifish. The first test, the optokinetic response (OKR), measures the reflexive eye movement triggered by motion in the visual field and allows assessment of visual acuity. The second assay, the dorsal light reflex (DLR), evaluates the swimming angle based on input of light coming from above. The OKR can be used to study the effect of aging on visual acuity as well as visual improvement and recovery after rejuvenation therapy or visual system injury or disease, whereas the DLR is best used to assess functional repair after a unilateral optic nerve crush.

Supplemental Digital Content is available in the text.

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system remains elusive. In the quest for neurostimulatory agents that promote regeneration, well-defined models and analysis methods are required. Tissue explant cultures closely resemble the in vivo situation, making them ideal to study the effect of compounds on the neuro-glial network. This study reports the optimization of an explant culture technique using retinas of neonatal mice and the development of an analysis script that allows for rapid and automated analysis of neurite outgrowth from these explants. The key features of this script (i.e., local thresholding and form selection) allow for swift and unbiased detection of neurite outgrowth. The novel analysis method is compared with two commonly used manual methods and successfully validated by performing dose-response studies with molecules known to either inhibit (anti–β1-integrin antibody) or stimulate (brain-derived neurotrophic factor and ciliary neurotrophic factor) neurite outgrowth from retinal explants. Finally, the new analysis script is used to study whether retinal explant origin has any effect on neurite outgrowth.

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)–2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)–2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

Growth arrest-specific gene 6 (Gas6) belongs to the family of vitamin K-dependent coagulation proteins, but in contrast to its other members, has only a limited role in hemostasis. Instead, Gas6 plays a prominent role in conditions of injury, inflammation and repair. Gas6 amplifies the activation of various cell types including endothelial cells and platelets in different models of thrombosis and inflammation, processes also important in atherosclerosis.

In preterms, low serum levels of IGF (IGF-I) correlate with retinopathy of prematurity (ROP). In mice, IGF-I is a prerequisite for normal retinal development. We further explored the link between IGF-I and oxygen-induced retinopathy (OIR). To assess the role of endogenous IGF-I, pups were redistributed into smaller versus larger litters at birth; in one subgroup, we measured body weight and circulating IGF-I; in another, we applied hyperoxia and assessed retinal neovascularization (NV). To screen for the potential role of exogenous IGF-I, we administered a single bolus of rhIGF-I on postnatal day (P) 4 to pups in normal litters, and applied hyperoxia; body weight and IGF-I were measured; maturation and NV were assessed. Neonatal mice in larger litters had a lower body weight than mice in smaller litters; they had lower levels of circulating IGF-I, and developed more OIR (p = 0.002). Mice who had received rhIGF-I, weighed more and had higher endogenous IGF-I levels; they matured faster and developed less OIR (p = 0.00001). These findings in mice are the first to support the notion that higher availability of endogenous or exogenous IGF-I reduces OIR risk, and thus sharpen the perspective that ROP may be preventable by briefly up-regulating IGF-I after birth.

In preterms, low serum levels of IGF (IGF-I) correlate with retinopathy of prematurity (ROP). In mice, IGF-I is a prerequisite for normal retinal development. We further explored the link between IGF-I and oxygen-induced retinopathy (OIR). To assess the role of endogenous IGF-I, pups were redistributed into smaller versuslarger litters at birth; in one subgroup, we measured body weight and circulating IGF-I; in another, we applied hyperoxia and assessed retinal neovascularization (NV). To screen for the potential role of exogenous IGF-I, we administered a single bolus of rhIGF-I on postnatal day (P) 4 to pups in normal litters, and applied hyperoxia; body weight and IGF-I were measured; maturation and NV were assessed. Neonatal mice in larger litters had a lower body weight than mice in smaller litters; they had lower levels of circulating IGF-I, and developed more OIR (p0.002). Mice who had received rhIGF-I, weighed more and had higher endogenous IGF-I levels; they matured faster and developed less OIR (p0.00001). These findings in mice are the first to support the notion that higher availability of endogenous or exogenous IGF-I reduces OIR risk, and thus sharpen the perspective that ROP may be preventable by briefly up-regulating IGF-I after birth.

The importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevis tadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of xVEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of xVEGF-D with xVEGF-C at suboptimal morpholino concentrations resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or xVEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.

The importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevistadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of xVEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of xVEGF-D with xVEGF-C at suboptimal morpholino concentrations resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or xVEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.

The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.

The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.

We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivinlox/loxmice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.

We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivinlox/lox mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.

Smads are intracellular signaling proteins that transduce signals elicited by members of the transforming growth factor (TGF)-β superfamily. Smad5 and Smad1 are highly homologous, and they mediate primarily bone morphogenetic protein (Bmp) signals. We used the Cre-loxPsystem and Sm22-Creand Tie-1-Cremice to study the function of Smad5 in the developing blood vessel wall. Analysis of embryos demonstrated that deletion of Smad5in endothelial or smooth muscle cells resulted in a normal organization of embryonic and extra-embryonic vasculature. Angiogenic assays performed in adult mice revealed that mutant mice display a comparable angiogenic and vascular remodeling response to control mice. In Sm22-Cre;Smad5fl/−mice, Smad5is also deleted in cardiomyocytes. Echocardiographic analysis on those 9-month-old female mice demonstrated larger left ventricle internal diameters and decreased fractional shortening compared with control littermates without signs of cardiac hypertrophy. The decreased cardiac contractility was associated with a decreased performance in a treadmill experiment. In isolated cardiomyocytes, fractional shortening was significantly reduced compared with control cells. These data demonstrate that restricted deletion of Smad5in the blood vessel wall results in viable mice. However, loss of Smad5 in cardiomyocytes leads to a mild heart defect.

Left ventricular (LV) hypertrophy is a natural response of the heart to increased pressure loading, but accompanying fibrosis and dilatation may result in irreversible life-threatening heart failure. Matrix metalloproteinases (MMPs) have been invoked in various cardiac diseases, however, direct genetic evidence for a role of the plasminogen activator (PA) and MMP systems in pressure overload-induced LV hypertrophy and in heart failure is lacking. Therefore, the consequences of transverse aortic banding (TAB) were analyzed in mice lacking tissue-type PA (t-PA−/−), urokinase-type PA (u-PA−/−), or gelatinase-B (MMP-9−/−), and in wild-type (WT) mice after adenoviral gene transfer of the PA-inhibitor PAI-1or the MMP-inhibitor TIMP-1. TAB elevated LV pressure comparably in all genotypes. In WT and t-PA−/−mice, cardiomyocyte hypertrophy was associated with myocardial fibrosis, LV dilatation and dysfunction, and pump failure after 7 weeks. In contrast, in u-PA−/−mice or in WT mice after PAI-1-and TIMP-1-gene transfer, cardiomyocyte hypertrophy was moderate and only minimally associated with cardiac fibrosis and LV dilatation, resulting in better preservation of pump function. Deficiency of MMP-9had an intermediate effect. These findings suggest that the use of u-PA- or MMP-inhibitors might preserve cardiac pump function in LV pressure overloading.

High-titer self-inactivating human immunodeficiency virus type-1 (HIV-1)–based vectors expressing the green fluorescent protein reporter gene that contained the central polypurine and termination tract and the woodchuck hepatitis virus posttranscriptional regulatory element were constructed. Transduction efficiency and biodistribution were determined, following systemic administration of these improved lentiviral vectors. In adult severe combined immunodeficiency (SCID) mice, efficient stable gene transfer was achieved in the liver (8.0% ± 6.0%) and spleen (24% ± 3%). Most transduced hepatocytes and nonhepatocytes were nondividing, thereby obviating the need to induce liver cell proliferation. In vivo gene transfer with this improved lentiviral vector was relatively safe since liver enzyme concentration in the plasma was only moderately and transiently elevated. In addition, nondividing major histocompatibility complex class II–positive splenic antigen-presenting cells (APCs) were efficiently transduced in SCID and normal mice. Furthermore, B cells were efficiently transduced, whereas T cells were refractory to lentiviral transduction in vivo. However, in neonatal recipients, lentiviral transduction was more widespread and included not only hepatocytes and splenic APCs but also cardiomyocytes. The present study suggests potential uses of improved lentiviral vectors for gene therapy of genetic blood disorders resulting from serum protein deficiencies, such as hemophilia, and hepatic disease. However, the use of liver-specific promoters may be warranted to circumvent inadvertent transgene expression in APCs. In addition, these improved lentiviral vectors could potentially be useful for genetic vaccination and treatment of perinatal cardiac disorders.

High-titer self-inactivating human immunodeficiency virus type-1 (HIV-1)–based vectors expressing the green fluorescent protein reporter gene that contained the central polypurine and termination tract and the woodchuck hepatitis virus posttranscriptional regulatory element were constructed. Transduction efficiency and biodistribution were determined, following systemic administration of these improved lentiviral vectors. In adult severe combined immunodeficiency (SCID) mice, efficient stable gene transfer was achieved in the liver (8.0% ± 6.0%) and spleen (24% ± 3%). Most transduced hepatocytes and nonhepatocytes were nondividing, thereby obviating the need to induce liver cell proliferation. In vivo gene transfer with this improved lentiviral vector was relatively safe since liver enzyme concentration in the plasma was only moderately and transiently elevated. In addition, nondividing major histocompatibility complex class II–positive splenic antigen-presenting cells (APCs) were efficiently transduced in SCID and normal mice. Furthermore, B cells were efficiently transduced, whereas T cells were refractory to lentiviral transduction in vivo. However, in neonatal recipients, lentiviral transduction was more widespread and included not only hepatocytes and splenic APCs but also cardiomyocytes. The present study suggests potential uses of improved lentiviral vectors for gene therapy of genetic blood disorders resulting from serum protein deficiencies, such as hemophilia, and hepatic disease. However, the use of liver-specific promoters may be warranted to circumvent inadvertent transgene expression in APCs. In addition, these improved lentiviral vectors could potentially be useful for genetic vaccination and treatment of perinatal cardiac disorders.

The role of the cytosolic domain of tissue factor (TF) in signal transduction and gene regulation was studied in mice with a targeted deletion of the 18 carboxy-terminal intracellular amino acids. This deletion was introduced in exon 6 along with a floxed neoR selection cassette in intron 5 using homologous recombination in embryonic stem cells. Removal of the floxed neoR cassette by in vivo Cre-mediated loxP recombination yielded TF+/δCT and TFδCT/δCT mice. In contrast to TF−/− mice, TF+/δCT and TFδCT/δCT mice displayed normal embryonic development, survival, fertility, and blood coagulation. Factor VIIa or factor Xa stimulation produced similar p44/42 MAPK activation in TF+/+ and TFδCT/δCT fibroblasts. These data, based on expression of a TFδCT molecule from the endogenous TF locus, provide conclusive proof that the cytosolic domain of TF is not essential for signal transduction in embryogenesis and in physiological postnatal processes.

The plasminogen system plays an important role in the proteolytic degradation of extracellular matrices during wound healing. In the present study we investigated the impact of the plasminogen system on cardiac wound healing and function after myocardial infarction. Myocardial infarction was induced in plasminogen-deficient mice (Plg−/−) and in wild-type controls (Plg+/+). Structural analysis 1, 2, and 5 weeks after infarction revealed that infarct healing was virtually abolished in Plg−/− mice, indicating that the plasminogen system is required for the repair process of the heart after infarction. In the absence of plasminogen, inflammatory cells did not migrate into the infarcted myocardium. Necrotic cardiomyocytes were not removed and the formation of granulation tissue and fibrous tissue did not occur. In these non-healing infarcted hearts, LV dilatation was not altered. In addition, gelatinolytic activity of MMP-2 and MMP-9 was depressed in the Plg−/− infarcted hearts, suggesting that the plasmin effect on infarct healing may be mediated by MMPs. Surprisingly, cardiac function was only attenuated to a rather small extent in the Plg−/− infarcted mice when compared to the wild-types. This study provides direct prove that plasmin-mediated proteolysis plays a central role in cardiac wound healing after myocardial infarction in mice.

Previous light microscopic studies have revealed neuropeptide-immunoreactive neurosecretory fibers in the teleostean neurohypophysis, and ultrastructural work has reported direct innervation of endocrine cells by the terminals of fibers penetrating the adenohypophysis. This paper reviews our recent data from ultrastructural, immunohistochemical, receptor localization, and superfusion studies, which suggest a role for neuropeptides in the control of teleost pituitary secretion. We have used a combination of pre- and post-embedding electron microscopic immunolabeling methods to determine which neuropeptides are present in fibers innervating the pituitaries of three species: Poecilia latipinna, Dicentrarchus labrax, and Clarias gariepinus. Numerous axon profiles with immunoreactivity for the neurosecretory peptides vasotocin and isotocin formed large Herring bodies and terminal-like boutons in contact with corticotropic, growth hormone, thyrotropic, and pars intermedia cells. Numerous melanin-concentrating hormone-immunoreactive fibers and scarcer neurotensin and corticotropin-releasing factor-immunoreactive fibers showed similar distributions, terminating close to pars intermedia and corticotropic cells. Somatostatin, cholecystokinin, galanin, substance P, neuropeptide Y, growth hormone-releasing factor, thyrotropin-releasing hormone, and gonadotropin-releasing hormone-immunoreactivities were found in small calibre fibers penetrating among growth hormone, thyrotropic, and gonadotropic cells. These morphological findings have been supplemented by autoradiographic studies, which showed the distribution of binding sites for vasotocin, isotocin, galanin, and neuropeptide Y ligands over specific groups of pituitary cells, and superfusion studies that showed growth hormone release was stimulated by growth hormone-releasing factor and thyrotropin-releasing hormone, but inhibited by somatostatin. The implications of these results for neuropeptidergic control of teleostean pituitary secretions are discussed. Microsc. Res. Tech. 44:19–35, 1999. © 1999 Wiley-Liss, Inc.

Previous light microscopic studies have revealed neuropeptide‐immunoreactive neurosecretory fibers in the teleostean neurohypophysis, and ultrastructural work has reported direct innervation of endocrine cells by the terminals of fibers penetrating the adenohypophysis. This paper reviews our recent data from ultrastructural, immunohistochemical, receptor localization, and superfusion studies, which suggest a role for neuropeptides in the control of teleost pituitary secretion. We have used a combination of pre‐ and post‐embedding electron microscopic immunolabeling methods to determine which neuropeptides are present in fibers innervating the pituitaries of three species: Poecilia latipinna, Dicentrarchus labrax,and Clarias gariepinus.Numerous axon profiles with immunoreactivity for the neurosecretory peptides vasotocin and isotocin formed large Herring bodies and terminal‐like boutons in contact with corticotropic, growth hormone, thyrotropic, and pars intermedia cells. Numerous melanin‐concentrating hormone‐immunoreactive fibers and scarcer neurotensin and corticotropin‐releasing factor‐immunoreactive fibers showed similar distributions, terminating close to pars intermedia and corticotropic cells. Somatostatin, cholecystokinin, galanin, substance P, neuropeptide Y, growth hormone‐releasing factor, thyrotropin‐releasing hormone, and gonadotropin‐releasing hormone‐immunoreactivities were found in small calibre fibers penetrating among growth hormone, thyrotropic, and gonadotropic cells. These morphological findings have been supplemented by autoradiographic studies, which showed the distribution of binding sites for vasotocin, isotocin, galanin, and neuropeptide Y ligands over specific groups of pituitary cells, and superfusion studies that showed growth hormone release was stimulated by growth hormone‐releasing factor and thyrotropin‐releasing hormone, but inhibited by somatostatin. The implications of these results for neuropeptidergic control of teleostean pituitary secretions are discussed. Microsc. Res. Tech. 44:19–35, 1999. © 1999 Wiley‐Liss, Inc.

The molecular mechanisms predisposing to atherosclerotic aneurysm formation remain undefined%1,5. Nevertheless, rupture of aortic aneurysms is a major cause of death in Western societies, with few available treatments and poor long-term progno–sis. Indirect evidence suggests that matrix metalloproteinases (MMPs) and plasminogen activators (PAs) are involved in its pathogenesis%1,6–12. MMPs are secreted as inactive zymogens (pro-MMPs), requiring activation in the extracellular compartment%11,13. Plasmin, generated from the zymogen plasminogen by tissue-type plasminogen activator (t-PA) or urokinase-type plas-minogen activator (u-PA; refs 14,15), has been proposed as a pos–sible activator in vitro, but evidence for such a role in vivo is lacking%16,17. Analysis of atherosclerotic aorta in mice with a deficiency of apoliprotein E (Apoe−/− ref. 18), singly or combined with a deficiency of t-PA (Apoe−/−.PIar−/−) or of u-PA (Apoe−/−-iPlair−/−, ref. 19), indicated that deficiency of u-PA protected against media destruction and aneurysm formation, probably by means of reduced plasmin-dependent activation of pro-MMPs. This genetic evidence suggests that plasmin is a pathophysiologically signifi–cant activator of pro-MMPs in vivo and may have implications for the design of therapeutic strategies to prevent aortic-wall destruction by controlling Plau gene function.

ABSTRACTIn this study, we investigated the potential antifungal activity of the alkylphospholipid oleylphosphocholine (OlPC), a structural analogue of miltefosine, on in vitroand in vivoCandida albicansbiofilm formation. The effect of OlPC on in vitroand in vivoC. albicansbiofilms inside triple-lumen polyurethane catheters was studied. In vivobiofilms were developed subcutaneously after catheter implantation on the lower back of Sprague-Dawley rats. Animals were treated orally with OlPC (20 mg/kg of body weight/day) for 7 days. The effect of OlPC on biofilms that developed on the mucosal surface was studied in an ex vivomodel of oral candidiasis. The role of OlPC in C. albicansmorphogenesis was investigated by using hypha-inducing media, namely, Lee, Spider, and RPMI 1640 media. OlPC displayed activity against both planktonic cells and in vitroC. albicansbiofilms. To completely abolish preformed, 24-h-old biofilms, higher concentrations (8, 10, and 13 mg/liter) were needed. Moreover, OlPC was able to reduce C. albicansbiofilms formed by caspofungin-resistant clinical isolates and acted synergistically when combined with caspofungin. The daily oral administration of OlPC significantly reduced in vivoC. albicansbiofilms that developed subcutaneously. In addition, OlPC decreased biofilm formation on mucosal surfaces. Interestingly, the application of subinhibitory concentrations of OlPC already inhibited the yeast-to-hypha transition, a crucial virulence factor of C. albicans. We document, for the first time, the effects of OlPC on C. albicanscells and suggest the potential use of OlPC for the treatment of C. albicansbiofilm-associated infections.

We previously identified the plasmin protease family as a critical determinant of the mobilization of hematopoietic stem and progenitor cells (HSC/HPC), but the role of the urokinase receptor uPAR remained unclear. uPAR is a membrane-anchored glycoprotein, which not only localizes its ligand urokinase (uPA) to the cell surface via its GPI-anchor but also regulates β1-integrin dependent cell adhesion and migration. Following 5-FU myeloablation or G-CSF treatment, mice lacking uPAR (uPAR−/−) had impaired hematopoietic recovery and HSC/HPC mobilization as compared to wild type (WT) mice. However, this phenotype was not mimicked in mice lacking uPA, suggesting a role of uPAR in mobilization independent of uPA-mediated proteolysis. The impaired mobilization in uPAR−/− mice was reversed upon pre-transplantation with WT BM cells (BMC), suggesting functional expression of uPAR on transplantable BMCs. Conversely, loss or inhibition of uPAR on transplanted BMCs impaired homing to the BM but not to the spleen, and compromised survival of myeloablated WT recipients. In vitro experiments revealed that loss or inhibition of uPAR impaired BMC adhesion to stromal cells and fibronectin. Anti-α4-β1 antibodies blocked adhesion of WT but not uPAR−/− BMCs. Thus, uPAR appears to regulate BM homing and α4-β1 dependent retention of transplantable BMCs, possibly HSC/HPCs. If uPAR mediates retention of HSC/HPCs, then this signal should be inactivated upon mobilization. Indeed, in 5-FU or G-CSF-treated WT mice, we found increased uPAR cleavage, and elevated levels of soluble uPAR (suPAR) in BM plasma. These processes failed to occur in mice lacking plasminogen, suggesting that plasmin cleaves uPAR during mobilization. Cleavage of uPAR appeared critical as the inactivation of the retention signals membrane-bound Kit ligand and SDF-1α was normal in uPAR−/− mice. Moreover, the generated suPAR may also affect the BM, as administration of recombinant suPAR in WT mice enhanced hematopoietic recovery and HSC/HPC mobilization after 5-FU or G-CSF. In vitro and transplantation experiments revealed that suPAR blocked α4-β1 dependent adhesion. Thus, in steady state, membrane-anchored uPAR appears to function as a BM retention signal for transplantable BMCs, possibly HSC/HPCs. In conditions of mobilization, the uPAR retention signal is cleaved, which weakens α4-β1 dependent adhesion and allows mobilization out of the BM. Soluble uPAR may then additionally amplify mobilization, in part by further attenuating α4-β1 dependent adhesion to the BM. Currently, we are investigating the role of uPAR on subsets of HSC/HPCs, and in the different BM niches. We are also performing long-term competitive repopulation experiments to further delineate the therapeutic potential of uPAR.

We previously identified the plasmin protease family as a critical determinant of the mobilization of hematopoietic stem and progenitor cells (HSC/HPC), but the role of the urokinase receptor uPAR remained unclear. uPAR is a membrane-anchored glycoprotein, which not only localizes its ligand urokinase (uPA) to the cell surface via its GPI-anchor but also regulates β1-integrin dependent cell adhesion and migration.

Hematopoietic stem/progenitor cells (HSC/HPCs) are retained in the bone marrow (BM) niche via receptor-ligand interactions and mobilized from the BM after proteolytic degradation of these retention complexes. Yet, the proteinases and retention signals involved remain incompletely identified. Here, we studied the role of the plasminogen proteinase system with its plasminogen activators PA) tPA and uPA, and active plasmin (Pli) in chemo- and G-CSF-induced mobilization of HPCs and HSCs. Therefore, 5-fluorouracil (5-FU) or G-CSF were administered to mice lacking plasminogen (Plg−/−), tPA (tPA−/−), uPA (uPA−/−), both activators (tPA−/−uPA−/−), uPAR (uPAR−/−), PAI-1 (PAI-1−/−), or α2-antiplasmin (α2-AP−/−). 5-FU treatment in WT mice elevated Pli activity in BM plasma 5-fold, killed 10% of WT mice, and increased number/proliferation of HSC/HPCs in the surviving mice, with full hematopoietic recovery after 3 weeks. In contrast, up to 75% of 5-FU-treated Plg−/−and tPA−/−uPA−/− demised in the early phase of recovery, with reduced number/proliferation of HSC/HPCs and delayed hematopoietic recovery. Following G-CSF, deficiency of Plg or inhibition of Pli by tranexamic acid reduced HPC expansion and HSC translocation in the BM, resulting in impaired HPC/HSC mobilization, by up to 55% and 75%, respectively. Further analysis using uPA−/− and tPA−/− mice revealed that uPA was critical for 5-FU-induced mobilization, whereas tPA was crucial for G-CSF-induced mobilization. In addition, analysis of mice lacking MMP-2, -3, -9, and -12 revealed that 5-FU- and G-CSF-induced mobilization required predominantly MMP-9 and -3, respectively. MMP-3 and MMP-9 activities upon mobilization were reduced in Plg−/− mice, suggesting that Pli activates these MMPs. In the absence of Plg, degradation of fibronectin in the BM and production of soluble Kit ligand (but not SDF1 α) were impaired, indicating additional downstream targets of Pli. uPAR is a membrane-anchored receptor for uPA, which is cleaved into a soluble form (suPAR) by Pli and other proteinases. Interestingly, uPAR was expressed on BM-derived HPC/HSCs, and uPAR deficiency reduced their retention within the BM niche in vitro and in vivo. Furthermore, uPAR−/− mice showed poor HSC/HPC mobilization in response to 5-FU and G-CSF, while suPAR administration in WT mice amplified G-CSF-induced mobilization. Moreover, suPAR levels in the BM were increased in WT but not in Plg−/− mice during mobilization, indicating, all together, that uPAR might be a novel retention signal for HSC/HPCs in the BM niche. Finally, increased Pli activity in PAI-1−/− and α2-AP−/− mice, and WT mice treated with tenecteplase (i.e. recombinant tPA variant used for clinical thrombolysis) enhanced G-CSF-induced mobilization. Importantly, initial results suggest that thrombolytic treatment of individuals after acute myocardial infarction also seemed to stimulate HPC mobilization, extrapolating our findings to man. In conclusion, these genetic and pharmacological data reveal, for the first time, a novel role for uPAR as a retention signal for HSC/HPCs in the BM and suggest that strategies to increase PA or Pli activity might offer novel therapeutic opportunities for HSC/HPC mobilization.

Hematopoietic stem/progenitor cells (HSC/HPCs) are retained in the bone marrow (BM) niche via receptor-ligand interactions and mobilized from the BM after proteolytic degradation of these retention complexes. Yet, the proteinases and retention signals involved remain incompletely identified. Here, we studied the role of the plasminogen proteinase system with its plasminogen activators PA) tPA and uPA, and active plasmin (Pli) in chemo- and G-CSF-induced mobilization of HPCs and HSCs.

The development of efficient transgenic technologies in mice has allowed the study of the consequences of genetic alterations on cardiovascular (patho)physiology, although the development of such models have been hampered by size limitation of species resulting in time-consuming, labor-intensive and costly analyses. This overview summarizes the murine models currently available for studying or manipulating angiogenesis, arterial stenosis, atherosclerosis, transplant arteriopathy, thrombosis, thrombolysis and bleeding and addresses techniques to evaluate vascular development during embryogenesis.