Hannes C. A. Drexler

Leptin, the Product of Ob Gene, Promotes Angiogenesis

The adipocyte-derived cytokine leptin is thought to play a key role in the control of satiety and energy expenditure. Because adipogenesis and angiogenesis are tightly correlated during the fat mass development, we tested the hypothesis that leptin is able to modulate the growth of the vasculature. Experiments were performed using cultured human umbilical venous endothelial cells (HUVECs) and porcine aortic endothelial cells. The presence of 170-kDa endothelial leptin receptor (Ob-R) was assessed in HUVECs by Western blot analysis. Reverse transcriptase-polymerase chain reaction analysis using specific oligonucleotides for the short and long Ob-R forms further revealed the expression of both Ob-R transcripts in endothelial cells. Moreover, leptin evoked a time-dependent tyrosine phosphorylation of a number of endothelial proteins, the most prominent of which were the mitogen-activated protein kinases Erk1/2. Treatment of HUVECs with leptin led to a concentration-dependent increase in cell number that was maximal at 10 ng/mL leptin and equivalent to that elicited by vascular endothelial growth factor. This effect was associated with an enhanced formation of capillary-like tubes in an in vitro angiogenesis assay and neovascularization in an in vivo model of angiogenesis. These results indicate that leptin, via activation of the endothelial Ob-R, generates a growth signal involving a tyrosine kinase-dependent intracellular pathway and promotes angiogenic processes. We speculate that this leptin-mediated stimulation of angiogenesis might represent not only a key event in the settlement of obesity but also may contribute to the modulation of growth under physiological and pathophysiological conditions in other tissues.

Myocytes Die by Multiple Mechanisms in Failing Human Hearts

&NA; We tested the hypothesis that myocyte loss in failing human hearts occurs by different mechanisms: apoptosis, oncosis, and autophagic cell death. Explanted hearts from 19 patients with idiopathic dilated cardiomyopathy (EF≤20%) and 7 control hearts were analyzed. Myocyte apoptosis revealed by caspase‐3 activation and TUNEL staining occurred at a rate of 0.002±0.0005% (P<0.05 versus control) and oncosis assessed by complement 9 labeling at 0.06±0.001% (P<0.05). Cellular degeneration including appearance of ubiquitin containing autophagic vacuoles and nuclear disintegration was present at the ultrastructural level. Nuclear and cytosolic ubiquitin/protein accumulations occurred at 0.08±0.004% (P<0.05). The ubiquitin‐activating enzyme E1 and the ligase E3 were not different from control. In contrast, ubiquitin mRNA levels were 1.8‐fold (P<0.02) elevated, and the conjugating enzyme E2 was 2.3‐fold upregulated (P<0.005). The most important finding, however, is the 2.3‐fold downregulation of the deubiquitination enzyme isopeptidase‐T and the 1.5‐fold reduction of the ubiquitin‐fusion degradation system‐1, which in conjunction with unchanged proteasomal subunit levels and proteasomal activity results in massive storage of ubiquitin/protein complexes and in autophagic cell death. A 2‐fold decrease of cathepsin D might be an additional factor responsible for the accumulation of ubiquitin/protein conjugates. It is concluded that in human failing hearts apoptosis, oncosis, and autophagy act in parallel to varying degrees. A disturbed balance between a high rate of ubiquitination and inadequate degradation of ubiquitin/protein conjugates may contribute to autophagic cell death. Together, these different types of cell death play a significant role for myocyte disappearance and the development of contractile dysfunction in failing hearts. (Circ Res. 2003;92:715–724.)

Platelet-Derived Growth Factor is Angiogenic In Vivo

PDGF receptors have recently been found to be expressed in microvascular endothelium in vivo under circumstances of endothelial cell activation and angiogenesis suggesting that PDGF may have a direct effect on endothelial cells. We have tested the angiogenic activity of PDGF-AA and -BB homodimers in the chick chorioallantoic membrane in vivo. PDGF-BB was found to consistently induce an angiogenic response whereas PDGF-AA was less active. Morphological analyses revealed that there was little inflammation associated with this response but an increase in vessel density suggested a direct effect of PDGF on embryonic chorioallantoic endothelial cells. In vitro, PDGF-BB was found to be more potent than PDGF-AA in stimulating the chemotaxis of rat brain capillary endothelial cells. This is consistent with a direct effect of PDGF on endothelial cells. Thus, this novel angiogenic activity of PDGF has implications for several developmental and pathological events in which PDGF, particularly the B-chain, is expressed.

Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment

Mouse endothelioma cells expressing the polyoma middle T oncogene induced hemangiomas in a variety of species such as mice, rats, chicks, and quails. In embryos and newborn mice the hemangiomas expanded within 10-18 hr of injection, disrupting the vasculature and causing the death of the animal. In contrast, the hemangiomas formed a stable structure reminiscent of benign human hemangiomas in adult mice within 5 days. Analysis of the cells comprising the hemangioma revealed that over 95% of the endothelial cells were host derived. No induction of host cell proliferation was detected, and no endothelial mitogens were secreted by the endothelioma cells in vitro. The maintenance of the hemangioma appeared to require the continuous presence of endothelioma cells. The results indicate that these endothelioma cells act as a potent stimulating agent in the rapid formation of hemangiomas by recruiting nonproliferating host endothelial cells.

p38 MAP kinase—a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability

Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but also is a potent inducer of vascular permeability. Although a dissection of the molecular pathways between angiogenesis‐ and vascular permeability‐inducing properties would be desirable for the development of angiogenic and anti‐angiogenic therapies, such mechanisms have not been identified yet. Here we provide evidence for a role of the p38 MAPK as the signaling molecule that separates these two processes. Inhibition of p38 MAPK activity enhances VEGF‐induced angiogenesis in vitro and in vivo, a finding that was accompanied by prolonged Erk1/2 MAPK activation, increased endothelial survival, and plasminogen activation. Conversely, the same inhibitors abrogate VEGF‐induced vascular permeability in vitro and in vivo. These dualistic properties of p38 MAPK are relevant not only for therapeutic angiogenesis but also for reducing edema formation and enhancing tissue repair in ischemic diseases.

Spatial regulation of VEGF receptor endocytosis in angiogenesis

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.

Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells

Proteolysis mediated by the ubiquitin‐proteasome system has been implicated in the regulation of programmed cell death. Here we investigated the differential effects of proteasomal inhibitors on the viability of proliferating and quiescent primary endothelial cells in vitro and in vivo. Subconfluent, proliferating cells underwent carbobenzoxy‐L‐isoleucyl‐7‐í‐butyl‐L‐glutamyl‐L‐alanyl‐L‐leucinal (PSI)‐induced apoptosis at low concentrations (EC50=24 nM), whereas at least 340‐fold higher concentrations of PSI were necessary to obtain the same effect in confluent, contact‐inhibited cells. PSI‐mediated cell death could be blocked by a caspase‐3 inhibitor (Ac‐DEVD‐H), but not by a caspase‐1 inhibitor (Ac‐YVAD‐H), suggesting that a caspase‐3‐like enzyme is activated during PSI‐induced apoptosis. When applied to the embryonic chick chorioallantoic membrane, a rapidly expanding tissue, PSI induced massive apoptosis also in vivo. PSI treatment of the CAM led to the formation of areas devoid of blood flow due to the induction of apoptosis in endothelial and other cells and to the collapse of capillaries and first order vessels. Our results demonstrate that proteasomal inhibitors such as PSI may prove effective as novel anti‐angiogenic and anti‐neoplastic substances.—Drexler, H. C. A., Risau, W., Konerding, M. A. Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells. FASEB J. 14, 65–77(2000)

Regression of vessels in the tunica vasculosa lentis is initiated by coordinated endothelial apoptosis: a role for vascular endothelial growth factor as a survival factor for endothelium.

The development of the embryonic lens is dependent on the formation and regression of the tunica vasculosa lentis (TVL), which is a transiently occurring capillary plexus that surrounds the posterior part of the lens. In this study, by using the terminal deoxy‐nucleotidyl transferase mediated nick end‐labelling technique (TUNEL), electron microscopy, radioactive end‐labelling of DNA extracted from TVL, and the Comet assay, we show that widespread apoptosis of the endothelial cells that constitute the TVL is occurring already at embryonic day 17.5 (E17.5) of mouse development, much earlier than was reported previously (Jack [1972a] Am. J. Ophthalmol. 74:261–272; Lang [1997] Cell Death Diff. 4:12–20). In addition to apoptotic cell death, regression of this structure is associated with loss of capillary integrity, leakage of erythrocytes into the vitreal compartment, and phagocytosis of the apoptotic endothelium by tissue macrophages (hyalocytes).

FRITZ : A SECRETED FRIZZLED-RELATED PROTEIN THAT INHIBITS WNT ACTIVITY

Signaling molecules of the Wnt gene family are involved in the regulation of dorso-ventral, segmental and tissue polarity in Xenopus and Drosophila embryos. Members of the frizzled gene family, such as Drosophila frizzled-2 and rat frizzled-1, have been shown encode Wnt binding activity and to engage intracellular signal transduction molecules known to be part of the Wnt signaling pathway. Here we describe the cloning and characterization of Fritz, a mouse (mfiz) and human (hfiz) gene which codes for a secreted protein that is structurally related to the extracellular portion of the frizzled genes from Drosophila and vertebrates. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. We propose a model in which Fritz can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites.

A unique Oct4 interface is crucial for reprogramming to pluripotency

Terminally differentiated cells can be reprogrammed to pluripotency by the forced expression of Oct4, Sox2, Klf4 and c-Myc. However, it remains unknown how this leads to the multitude of epigenetic changes observed during the reprogramming process. Interestingly, Oct4 is the only factor that cannot be replaced by other members of the same family to induce pluripotency. To understand the unique role of Oct4 in reprogramming, we determined the structure of its POU domain bound to DNA. We show that the linker between the two DNA-binding domains is structured as an α-helix and exposed to the protein’s surface, in contrast to the unstructured linker of Oct1. Point mutations in this α-helix alter or abolish the reprogramming activity of Oct4, but do not affect its other fundamental properties. On the basis of mass spectrometry studies of the interactome of wild-type and mutant Oct4, we propose that the linker functions as a protein–protein interaction interface and plays a crucial role during reprogramming by recruiting key epigenetic players to Oct4 target genes. Thus, we provide molecular insights to explain how Oct4 contributes to the reprogramming process.