Michael Potente

Basic and Therapeutic Aspects of Angiogenesis

Blood vessels form extensive networks that nurture all tissues in the body. Abnormal vessel growth and function are hallmarks of cancer and ischemic and inflammatory diseases, and they contribute to disease progression. Therapeutic approaches to block vascular supply have reached the clinic, but limited efficacy and resistance pose unresolved challenges. Recent insights establish how endothelial cells communicate with each other and with their environment to form a branched vascular network. The emerging principles of vascular growth provide exciting new perspectives, the translation of which might overcome the current limitations of pro- and antiangiogenic medicine.

MicroRNA-92a Controls Angiogenesis and Functional Recovery of Ischemic Tissues in Mice

Of Life, Limb, and a Small RNA Gene expression in mammals is controlled not only by proteins but by small noncoding RNAs called microRNAs. The involvement of these RNAs provides powerful clues about the molecular origins of human diseases and how they might be treated. Ischemic diseases arise from an inadequate blood supply. Bonauer et al. (p. 1710, published online 21 May) find that a specific microRNA that is expressed in the cells lining blood vessels (called miR-92a) functions to repress the growth of new blood vessels. MiR-92a probably acts through effects on expression of integrins, proteins involved in cell adhesion and migration. In mouse models in which an inadequate blood supply had caused damage either to heart or limb muscle, therapeutic inhibition of miR-92a led to an increase in blood vessel density in the damaged tissues and enhanced functional recovery. Inhibition of a microRNA that represses blood vessel growth enhances the recovery of tissue damaged by an inadequate blood supply. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to target messenger RNAs (mRNAs), leading to translational repression or degradation. Here, we show that the miR-17~92 cluster is highly expressed in human endothelial cells and that miR-92a, a component of this cluster, controls the growth of new blood vessels (angiogenesis). Forced overexpression of miR-92a in endothelial cells blocked angiogenesis in vitro and in vivo. In mouse models of limb ischemia and myocardial infarction, systemic administration of an antagomir designed to inhibit miR-92a led to enhanced blood vessel growth and functional recovery of damaged tissue. MiR-92a appears to target mRNAs corresponding to several proangiogenic proteins, including the integrin subunit alpha5. Thus, miR-92a may serve as a valuable therapeutic target in the setting of ischemic disease.

MicroRNA-34a regulates cardiac ageing and function

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

Endothelial adherens junctions control tight junctions by VE-cadherin-mediated upregulation of claudin-5.

Intercellular junctions mediate adhesion and communication between adjoining cells. Although formed by different molecules, tight junctions (TJs) and adherens junctions (AJs) are functionally and structurally linked, but the signalling pathways behind this interaction are unknown. Here we describe a cell-specific mechanism of crosstalk between these two types of structure. We show that endothelial VE-cadherin at AJs upregulates the gene encoding the TJ adhesive protein claudin-5. This effect requires the release of the inhibitory activity of forkhead box factor FoxO1 and the Tcf-4–β-catenin transcriptional repressor complex. Vascular endothelial (VE)-cadherin acts by inducing the phosphorylation of FoxO1 through Akt activation and by limiting the translocation of β-catenin to the nucleus. These results offer a molecular basis for the link between AJs and TJs and explain why VE-cadherin inhibition may cause a marked increase in permeability.

Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization

Forkhead box O (Foxo) transcription factors are emerging as critical transcriptional integrators among pathways regulating differentiation, proliferation, and survival, yet the role of the distinct Foxo family members in angiogenic activity of endothelial cells and postnatal vessel formation has not been studied. Here, we show that Foxo1 and Foxo3a are the most abundant Foxo isoforms in mature endothelial cells and that overexpression of constitutively active Foxo1 or Foxo3a, but not Foxo4, significantly inhibits endothelial cell migration and tube formation in vitro. Silencing of either Foxo1 or Foxo3a gene expression led to a profound increase in the migratory and sprout-forming capacity of endothelial cells. Gene expression profiling showed that Foxo1 and Foxo3a specifically regulate a nonredundant but overlapping set of angiogenesis- and vascular remodeling-related genes. Whereas angiopoietin 2 (Ang2) was exclusively regulated by Foxo1, eNOS, which is essential for postnatal neovascularization, was regulated by Foxo1 and Foxo3a. Consistent with these findings, constitutively active Foxo1 and Foxo3a repressed eNOS protein expression and bound to the eNOS promoter. In vivo, Foxo3a deficiency increased eNOS expression and enhanced postnatal vessel formation and maturation. Thus, our data suggest an important role for Foxo transcription factors in the regulation of vessel formation in the adult.

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD+-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.

Nrarp Coordinates Endothelial Notch and Wnt Signaling to Control Vessel Density in Angiogenesis

When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections.

FOXO-dependent expression of the proapoptotic protein Bim: pivotal role for apoptosis signaling in endothelial progenitor cells

Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization. Risk factors for coronary artery disease reduce the number of EPCs in humans. Since EPC apoptosis might be a potential mechanism to regulate the number of EPCs, we investigated the effects of oxidative stress and HMG‐CoA‐reductase inhibitors (statins) on EPC apoptosis. Atorvastatin, mevastatin, or VEGF prevented EPC apoptosis induced by H2O2. The antiapoptotic effect was reversed by inhibition of the PI3K/Akt pathway. Forkhead transcription factors (FOXO1, FOXO3a, FOXO4) exert proapoptotic effects and are phosphorylated and, thereby, inactivated by Akt. Therefore, we elucidated the involvement of forkhead transcription factors. Atorvastatin induced the phosphorylation of the predominant forkhead factor FOXO4 in EPCs. In addition, atorvastatin reduced the expression of the proapoptotic forkhead‐regulated protein Bim in a PI3K‐dependent manner. Consistently, overexpression of FOXO4 activated the Bim promoter as determined by reporter gene expression and stimulated the expression of Bim, resulting in an increased EPC apoptosis. Statins failed to prevent EPC apoptosis induced by overexpression of Bim or nonphosphorylatable FOXO4, suggesting that the protective effects of statins depend on this pathway. In summary, our results show that FOXO‐dependent expression of Bim plays a pivotal role for EPC apoptosis. Statins reduce oxidative stress‐induced EPC apoptosis, inactivate FOXO4, and down‐regulate Bim.

Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells

The regulation of acetylation is central for the epigenetic control of lineage-specific gene expression and determines cell fate decisions. We provide evidence that the inhibition of histone deacetylases (HDACs) blocks the endothelial differentiation of adult progenitor cells. To define the mechanisms by which HDAC inhibition prevents endothelial differentiation, we determined the expression of homeobox transcription factors and demonstrated that HoxA9 expression is down-regulated by HDAC inhibitors. The causal involvement of HoxA9 in the endothelial differentiation of adult progenitor cells is supported by the finding that HoxA9 overexpression partially rescued the endothelial differentiation blockade induced by HDAC inhibitors. Knockdown and overexpression studies revealed that HoxA9 acts as a master switch to regulate the expression of prototypical endothelial-committed genes such as endothelial nitric oxide synthase, VEGF-R2, and VE-cadherin, and mediates the shear stress–induced maturation of endothelial cells. Consistently, HoxA9-deficient mice exhibited lower numbers of endothelial progenitor cells and showed an impaired postnatal neovascularization capacity after the induction of ischemia. Thus, HoxA9 is regulated by HDACs and is critical for postnatal neovascularization.

FOXO1 couples metabolic activity and growth state in the vascular endothelium.

Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1–MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation.