Josef Priller

Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1

The therapeutic potential of placental growth factor (PlGF) and its receptor Flt1 in angiogenesis is poorly understood. Here, we report that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to vascular endothelial growth factor (VEGF). An antibody against Flt1 suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in autoimmune arthritis. Anti-Flt1 also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not attributable to reduced plaque neovascularization. Inhibition of VEGF receptor Flk1 did not affect arthritis or atherosclerosis, indicating that inhibition of Flk1-driven angiogenesis alone was not sufficient to halt disease progression. The anti-inflammatory effects of anti-Flt1 were attributable to reduced mobilization of bone marrow–derived myeloid progenitors into the peripheral blood; impaired infiltration of Flt1-expressing leukocytes in inflamed tissues; and defective activation of myeloid cells. Thus, PlGF and Flt1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.

Stroke-induced Immunodeficiency Promotes Spontaneous Bacterial Infections and Is Mediated by Sympathetic Activation Reversal by Poststroke T Helper Cell Type 1–like Immunostimulation

Infections are a leading cause of death in stroke patients. In a mouse model of focal cerebral ischemia, we tested the hypothesis that a stroke-induced immunodeficiency increases the susceptibility to bacterial infections. 3 d after ischemia, all animals developed spontaneous septicemia and pneumonia. Stroke induced an extensive apoptotic loss of lymphocytes and a shift from T helper cell (Th)1 to Th2 cytokine production. Adoptive transfer of T and natural killer cells from wild-type mice, but not from interferon (IFN)-γ–deficient mice, or administration of IFN-γ at day 1 after stroke greatly decreased the bacterial burden. Importantly, the defective IFN-γ response and the occurrence of bacterial infections were prevented by blocking the sympathetic nervous system but not the hypothalamo-pituitary-adrenal axis. Furthermore, administration of the β-adrenoreceptor blocker propranolol drastically reduced mortality after stroke. These data suggest that a catecholamine-mediated defect in early lymphocyte activation is the key factor in the impaired antibacterial immune response after stroke.

Experimental autoimmune encephalomyelitis repressed by microglial paralysis

Although microglial activation occurs in inflammatory, degenerative and neoplastic central nervous system (CNS) disorders, its role in pathogenesis is unclear. We studied this question by generating CD11b-HSVTK transgenic mice, which express herpes simplex thymidine kinase in macrophages and microglia. Ganciclovir treatment of organotypic brain slice cultures derived from CD11b-HSVTK mice abolished microglial release of nitrite, proinflammatory cytokines and chemokines. Systemic ganciclovir administration to CD11b-HSVTK mice elicited hematopoietic toxicity, which was prevented by transfer of wild-type bone marrow. In bone marrow chimeras, ganciclovir blocked microglial activation in the facial nucleus upon axotomy and repressed the development of experimental autoimmune encephalomyelitis. We conclude that microglial paralysis inhibits the development and maintenance of inflammatory CNS lesions. The microglial compartment thus provides a potential therapeutic target in inflammatory CNS disorders. These results validate CD11b-HSVTK mice as a tool to study the impact of microglial activation on CNS diseases in vivo.

Targeting gene-modified hematopoietic cells to the central nervous system: Use of green fluorescent protein uncovers microglial engraftment

Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood–brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.

Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease

Mononuclear phagocytic cells in the CNS used to be defined according to their anatomical location and surface marker expression. Recently, this concept has been challenged by the results of developmental and gene expression profiling studies that have used novel molecular biological tools to unravel the origin of microglia and to define their role as specialized tissue macrophages with long lifespans. Here, we describe how these results have redefined microglia and helped us to understand how different myeloid cell populations operate in the CNS based on their cell-specific gene expression signatures, distinct ontogeny and differential functions. Moreover, we describe the vulnerability of microglia to dysfunction and propose that myelomonocytic cells might be used in the treatment of neurological and psychiatric disorders that are characterized by primary or secondary microgliopathy.

Bone Marrow–Derived Progenitor Cells Modulate Vascular Reendothelialization and Neointimal Formation: Effect of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibition

Objective—Atherosclerosis and restenosis after vascular injury are both characterized by endothelial dysfunction, apoptosis, inappropriate endothelialization, and neointimal formation. Bone marrow–derived endothelial progenitor cells have been implicated in neovascularization, resulting in adult blood vessel formation. Despite the anticipated stem cell plasticity, the role of bone marrow–derived endothelial progenitor cells has not been clarified in vascular lesion development. Methods and Results—We investigated vascular lesion formation in mice after transplantation of bone marrow transfected by means of retrovirus with enhanced green fluorescent protein. Carotid artery injury was induced, resulting in neointimal formation. Fluorescence microscopy and immunohistological analysis revealed that bone marrow–derived progenitor cells are involved in reendothelialization of the vascular lesions. Treatment with rosuvastatin (20 mg/kg body wt per day), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, enhanced the circulating pool of endothelial progenitor cells, propagated the advent of bone marrow–derived endothelial cells in the injured vessel wall, and, thereby, accelerated reendothelialization and significantly decreased neointimal formation. Conclusions—Vascular lesion development initiated by endothelial cell damage is moderated by bone marrow–derived progenitor cells. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibition promotes bone marrow–dependent reendothelialization and diminishes vascular lesion development. These findings may help to establish novel pathophysiological concepts and therapeutic strategies in the treatment of various cardiovascular diseases.

Estrogen Increases Bone Marrow–Derived Endothelial Progenitor Cell Production and Diminishes Neointima Formation

Background Estrogens improve endothelial function and accelerate reendothelialization after vascular injury via largely unknown mechanisms. Bone marrow‐derived endothelial progenitor cells (EPCs) are thought to positively influence endothelialization, vascular repair, and angiogenesis. Methods and Results In mice subjected to sham operation, ovariectomy, or ovariectomy and estrogen replacement treatment, estrogen deficiency significantly decreased EPCs circulating in the peripheral blood and residing in the bone marrow, as well as EPCs that were in vitro expanded from spleen‐derived mononuclear cells. These effects were completely prevented by estrogen replacement. Human women with increased estrogen plasma concentrations also displayed profoundly increased levels of circulating EPCs. Estrogens increase EPC numbers through a decreased apoptosis rate, which is mediated via a caspase‐8‐dependent pathway. Estrogen deficiency increased neointima formation after carotid artery injury in mice, but this effect was diminished by estrogen replacement therapy. In mice transplanted with green fluorescent protein‐positive bone marrow, reendothelialization of injured vessel segments by bone marrow‐derived cells was decreased during estrogen deficiency and increased in response to estrogen treatment. Conclusions Estrogens increase numbers of EPCs by antiapoptotic effects leading to accelerated vascular repair and decreased neointima formation. (Circulation. 2003;107:3059‐3065.)

Neogenesis of cerebellar Purkinje neurons from gene-marked bone marrow cells in vivo

The versatility of stem cells has only recently been fully recognized. There is evidence that upon adoptive bone marrow (BM) transplantation (BMT), donor-derived cells can give rise to neuronal phenotypes in the brains of recipient mice. Yet only few cells with the characteristic shape of neurons were detected 1–6 mo post-BMT using transgenic or newborn mutant mice. To evaluate the potential of BM to generate mature neurons in adult C57BL/6 mice, we transferred the enhanced green fluorescent protein (GFP) gene into BM cells using a murine stem cell virus-based retroviral vector. Stable and high level long-term GFP expression was observed in mice transplanted with the transduced BM. Engraftment of GFP-expressing cells in the brain was monitored by intravital microscopy. In a long-term follow up of 15 mo post-BMT, fully developed Purkinje neurons were found to express GFP in both cerebellar hemispheres and in all chimeric mice. GFP-positive Purkinje cells were also detected in BM chimeras from transgenic mice that ubiquitously express GFP. Based on morphologic criteria and the expression of glutamic acid decarboxylase, the newly generated Purkinje cells were functional.

Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain.

Modern functional imaging techniques of the brain measure local hemodynamic responses evoked by neuronal activity. Capillary pericytes recently were suggested to mediate neurovascular coupling in brain slices, but their role in vivo remains unexplored. We used two-photon microscopy to study in real time pericytes and the dynamic changes of capillary diameter and blood flow in the cortex of anesthetized mice, as well as in brain slices. The thromboxane A2 analog, 9,11-dideoxy-9α,11α-methanoepoxy Prostaglandin F2α (U46619), induced constrictions in the vicinity of pericytes in a fraction of capillaries, whereas others dilated. The changes in vessel diameter resulted in changes in capillary red blood cell (RBC) flow. In contrast, during brief epochs of seizure activity elicited by local administration of the GABAA receptor antagonist, bicuculline, capillary RBC flow increased without pericyte-induced capillary diameter changes. Precapillary arterioles were the smallest vessels to dilate, together with penetrating and pial arterioles. Our results provide in vivo evidence that pericytes can modulate capillary blood flow in the brain, which may be important under pathological conditions. However, our data suggest that precapillary and penetrating arterioles, rather than pericytes in capillaries, are responsible for the blood flow increase induced by neural activity.

Distinct and Non-Redundant Roles of Microglia and Myeloid Subsets in Mouse Models of Alzheimer's Disease

Mononuclear phagocytes are important modulators of Alzheimers disease (AD), but the specific functions of resident microglia, bone marrow-derived mononuclear cells, and perivascular macrophages have not been resolved. To elucidate the spatiotemporal roles of mononuclear phagocytes during disease, we targeted myeloid cell subsets from different compartments and examined disease pathogenesis in three different mouse models of AD (APPswe/PS1, APPswe, and APP23 mice). We identified chemokine receptor 2 (CCR2)-expressing myeloid cells as the population that was preferentially recruited to β-amyloid (Aβ) deposits. Unexpectedly, AD brains with dysfunctional microglia and devoid of parenchymal bone marrow-derived phagocytes did not show overt changes in plaque pathology and Aβ load. In contrast, restriction of CCR2 deficiency to perivascular myeloid cells drastically impaired β-amyloid clearance and amplified vascular Aβ deposition, while parenchymal plaque deposition remained unaffected. Together, our data advocate selective functions of CCR2-expressing myeloid subsets, which could be targeted specifically to modify disease burden in AD.