Aernout Luttun

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.

Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.

Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA–/–) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA–/– mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.

Heterozygous Deficiency of PHD2 Restores Tumor Oxygenation and Inhibits Metastasis via Endothelial Normalization

A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

Placental growth factor and its receptor, vascular endothelial growth factor receptor-1: novel targets for stimulation of ischemic tissue revascularization and inhibition of angiogenic and inflammatory disorders

Summary.  In contrast to VEGF and its receptor VEGFR‐2, PlGF and its receptor VEGFR‐1 have been largely neglected and therefore their potential for therapy has not been previously explored. In this review, we describe the molecular properties of PlGF and VEGFR‐1 and how this translates into an important role for PlGF in the angiogenic switch in pathological angiogenesis, by interacting with VEGFR‐1 and synergizing with VEGF. PlGF was effective in the growth of new and stable vessels in cardiac and limb ischemia, through its action on different cell types (i.e. endothelial, smooth muscle and inflammatory cells and their precursors) that play a cardinal role in blood vessel formation. Accordingly, blocking its receptor VEGFR‐1 with monoclonal antibodies (anti‐VEGFR‐1 mAb), expressed on al these cell types, successfully attenuated blood vessel formation during cancer, ischemic retinopathy and rheumatoid arthritis. In addition, while blocking this receptor was effective in reducing inflammatory disorders like atherosclerosis and rheumatoid arthritis, blocking the anti‐angiogenic receptor VEGFR‐2 was without effect. This indicates that in the latter diseases the beneficial effects of anti‐VEGFR1 mAb were mainly due to its effect on inflammatory cells. Importantly, VEGFR‐1 was also present on hematopoietic stem/progenitor cells, the precursors of inflammatory cells. Thus, these preclinical studies show proof‐of‐principle that PlGF and VEGFR‐1 are promising therapeutic targets to treat angiogenesis and inflammation related disorders. Clinical trials will reveal whether this is also true for patients.

Isolation and Characterization of Kidney-Derived Stem Cells

Acute kidney injury is followed by regeneration of damaged renal tubular epithelial cells. The purpose of this study was to test the hypothesis that renal stem cells exist in the adult kidney and participate in the repair process. A unique population of cells that behave in a manner that is consistent with a renal stem cell were isolated from rat kidneys and were termed multipotent renal progenitor cells (MRPC). Features of these cells include spindle-shaped morphology; self-renewal for >200 population doublings without evidence for senescence; normal karyotype and DNA analysis; and expression of vimentin, CD90 (thy1.1), Pax-2, and Oct4 but not cytokeratin, MHC class I or II, or other markers of more differentiated cells. MRPC exhibit plasticity that is demonstrated by the ability of the cells to be induced to express endothelial, hepatocyte, and neural markers by reverse transcriptase-PCR and immunohistochemistry. The cells can differentiate into renal tubules when injected under the capsule of an uninjured kidney or intra-arterially after renal ischemia-reperfusion injury. Oct4 expression was seen in some tubular cells in the adult kidney, suggesting these cells may be candidate renal stem cells. It is proposed that MRPC participate in the regenerative response of the kidney to acute injury.

Vascular Progenitors: From Biology to Treatment

The identification of endothelial progenitor cells (EPCs) in the adult human has forced us to reconsider how new blood vessels grow in physiological and pathological conditions in the adult human. An important question in angiogenesis research is to what extent these progenitors functionally contribute to revascularization of ischemic tissues and and to what extent they can be used for therapeutic angiogenic cell transplantation. Even more challenging is the concept that hematopoietic and other bone-marrow-derived stem cells might be recruited in the context of ischemia to induce neovessel formation. This review discusses some of the recent insights and outstanding questions on EPCs, both from a biological and therapeutic perspective.

Placental Growth Factor (PlGF) and Its Receptor Flt‐1 (VEGFR‐1)

Abstract: Efforts to therapeutically stimulate or inhibit vessel growth have been primarily focused on vascular endothelial growth factor (VEGF) and its receptor VEGFR‐2 (Flk‐1), while little attention has been devoted to the therapeutic potential for angiogenic disorders of placental growth factor (PlGF), a VEGF family member, and its receptor VEGFR‐1 (Flt‐1). However, recent developments and insights could shift that focus to P1GF and Flt‐1. Indeed, PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to VEGF and did not cause side effects associated with VEGF, such as edema or hypotension. An anti‐Flt‐1 antibody suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in arthritis. The anti‐Flt‐1 antibody also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not due to reduced plaque neovascularization. The anti‐inflammatory effects of the anti‐Flt‐1 antibody were attributable to a reduced mobilization of bone marrow‐derived myeloid progenitors into the peripheral blood, a reduced mobilization/differentiation (and impaired infiltration) of Flt‐1‐expressing leukocytes into inflamed tissues, and a defective activation of myeloid cells. Thus, PlGF and Flt‐1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.

Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors

The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.

Soluble VEGF receptor Flt1: the elusive preeclampsia factor discovered?

The occurrence of seizures (eclampsia, from the Greek “eklampsis,” sudden flashing) has been a long-known and feared complication of pregnancy, often killing both mother and child. Preeclampsia, or the condition preceding full-blown eclampsia, affects up to 5% of pregnant women and is diagnosed by the onset of hypertension and proteinuria in the second trimester (1). Preeclampsia may eventually progress to glomerular malfunction, thrombocytopenia, liver and brain edema, and associated life-threatening seizures (2) (Figure ​(Figure1).1). Preeclampsia has been sometimes termed the “disease of theories,” as several models for its pathogenesis have been proposed. But, as of today, no satisfactory unifying hypothesis has emerged (1). The restricted occurrence of preeclampsia to humans and primates and the lack of a suitable animal model have hampered the understanding of its pathogenesis (3). In this issue of the JCI, S.E. Maynard et al. (4) report the novel insight that circulating levels of two angiogenic growth factors, VEGF and placental growth factor (PlGF), may play a more important role than previously believed. In particular, the authors propose that, in pregnant women with preeclampsia, the placenta produces elevated levels of the soluble fms-like tyrosine kinase 1 (sFlt1) receptor, which captures free VEGF and PlGF. As a result, the normal vasculature in the kidney, brain, lungs, and other organs is deprived of essential survival and maintenance signals and becomes dysfunctional (Figure ​(Figure1).1). As the authors show in their rodent model, this may lead to the development of hypertension and renal disease, reminiscent of preeclampsia in humans. In another study in this issue, V. Eremina et al. (5) provide additional evidence for a critical role of VEGF in renal disease during preeclampsia. These authors demonstrate that mice lacking one VEGF allele in renal podocytes develop the typical renal pathology found in pregnant women with preeclampsia. These studies therefore shed unprecedented light on the pathogenesis of preeclampsia and offer novel therapeutic opportunities for this disease. Figure 1 Hypothesis on the role of sFlt1 in preeclampsia. (a) During normal pregnancy, the uterine spiral arteries are infiltrated and remodeled by endovascular invasive trophoblasts, thereby increasing blood flow significantly in order to meet the oxygen and ...

Differentiation Potential of Human Postnatal Mesenchymal Stem Cells, Mesoangioblasts, and Multipotent Adult Progenitor Cells Reflected in Their Transcriptome and Partially Influenced by the Culture Conditions†‡§

Several adherent postnatal stem cells have been described with different phenotypic and functional properties. As many of these cells are being considered for clinical therapies, it is of great importance that the identity and potency of these products is validated. We compared the phenotype and functional characteristics of human mesenchymal stem cells (hMSCs), human mesoangioblasts (hMab), and human multipotent adult progenitor cells (hMAPCs) using uniform standardized methods. Human MAPCs could be expanded significantly longer in culture. Differences in cell surface marker expression were found among the three cell populations with CD140b being a distinctive marker among the three cell types. Differentiation capacity towards adipocytes, osteoblasts, chondrocytes, and smooth muscle cells in vitro, using established protocols, was similar among the three cell types. However, only hMab differentiated to skeletal myocytes, while only hMAPCs differentiated to endothelium in vitro and in vivo. A comparative transcriptome analysis confirmed that the three cell populations are distinct and revealed gene signatures that correlated with their specific functional properties. Furthermore, we assessed whether the phenotypic, functional, and transcriptome features were mediated by the culture conditions. Human MSCs and hMab cultured under MAPC conditions became capable of generating endothelial‐like cells, whereas hMab lost some of their ability to generate myotubes. By contrast, hMAPCs cultured under MSC conditions lost their endothelial differentiation capacity, whereas this was retained when cultured under Mab conditions, however, myogenic capacity was not gained under Mab conditions. These studies demonstrate that hMSCs, hMab, and hMAPCs have different properties that are partially mediated by the culture conditions. STEM CELLS 2011;29:871–882