Alexandre Trindade

More Sustainable Approaches for the Synthesis of N-Based Heterocycles† ‡

Centro de Quı́mica-Fı́sica Molecular (CQFM) and Institute of Nanosciences and Nanotechnology (IN), Departamento de Engenharia Quı́mica e Biológica, Instituto Superior Técnico, 1049-001 Lisboa, Portugal, REQUIMTE, Departamento de Quı́mica, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal, and iMed.UL, Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal

Overexpression of delta-like 4 induces arterialization and attenuates vessel formation in developing mouse embryos

The importance of Notch signaling pathway in the regulation of vascular development and angiogenesis is suggested by the expression of Notch receptors and ligands in vascular endothelial cells (ECs) and the observed vascular phenotypes in mutants of Notch receptors or ligands, especially Dll4. DLL4 is specifically expressed in arterial ECs during development, and haplo-insufficiency is embryonically lethal in mice. To address the role of Dll4 in vascular development, we produced mDll4 conditionally overexpressed transgenic mice that were crossed with constitutive recombinase cre lines. Double transgenic embryos displayed grossly enlarged dorsal aortae (DA) and died before embryonic day 10.5 (E10.5), showing a variable degree of premature arteriovenous fusion. Veins displayed ectopic expression of arterial markers. Other defects included reduced vascular sprouting, EC proliferation, and migration. mDll4 overexpression also inhibited VEGF signaling and increased fibronectin accumulation around the vessels. In vitro and in vivo studies of DLL4-FL (Dll4-full-length) in ECs recapitulate many of the mDll4 transgenics findings, including decreased tube formation, reduced vascular branching, fewer vessels, increased pericyte recruitment, and increased fibronectin expression. These results establish the role of Dll4 in arterial identity determination, and regulation of angiogenesis subject to dose and location.

Combination of Dll4/Notch and Ephrin-B2/EphB4 targeted therapy is highly effective in disrupting tumor angiogenesis.

BackgroundDll4/Notch and Ephrin-B2/EphB4 pathways play critical roles in tumor vessel development and maturation. This study evaluates the efficacy of the inhibition of both signaling pathways, alone and in combination, in reducing the growth of an autochthonous mouse tumor and assesses potential adverse effects.MethodsWe used the transgenic RIP1-Tag2 tumor model to study the effects of 1) inhibition of Dll4/Notch by either Dll4 allelic deletion or use of a soluble extracellular Dll4 (sDll4), 2) inhibition of Ephrin-B2/EphB4 signaling by a soluble extracellular EphB4 fused to albumin (sEphB4-Alb), and 3) inhibition of both pathways by sEphB4-Alb combined with either Dll4 allelic deletion or sDll4. To investigate adverse effects, we used inducible endothelial-specific Dll4 knock-out mice, treated with sEphB4-Alb, and carried out histopathological analysis.ResultsDll4 allele deletion or soluble Dll4 treatment resulted in increased tumor vessel density, reduced mural cell recruitment and vessel perfusion which resulted in reduced tumor size. The soluble EphB4 instead reduced vessel density and vessel perfusion, leading to reduction of tumor size. Greater efficacy was observed when sEphB4-Alb was combined with either Dll4 allele deletion or sDll4 in regards to tumor size, vessel perfusion and mural cell recruitment. Induced endothelial specific Dll4 loss-of-function caused hepatic vascular alterations, which were prevented by concomitant sEphB4-Alb treatment.ConclusionCombination targeting of Dll4/Notch and Ephrin-B2/EphB4 has potential for clinical investigation, providing cumulative efficacy and increased safety over Dll4/Notch inhibition alone.

Axial coordination of NHC ligands on dirhodium(II) complexes: Generation of a new family of catalysts

An efficient new methodology for the arylation of aldehydes is disclosed which uses dirhodium(II) catalysts and N-heterocyclic carbene (NHC) ligands. Complexes of Rh 2(OAc) 4 with one and two NHCs attached on the axial positions were successfully isolated, fully characterized, and used as catalysts in the reaction. The saturated monocomplex ((NHC 5)Rh 2(OAc) 4) 31 was shown to be the most active catalyst and was particularly efficient in the arylation of alkyl aldehydes. DFT calculations support participation of complexes with one axial NHC in the reaction as the catalysts active species and indicate that hydrogen bonds involving dirhodium unit, reactants, and solvent (alcohol) play an important role on the reaction mechanism.

Loss of Notch signalling induced by Dll4 causes arterial calibre reduction by increasing endothelial cell response to angiogenic stimuli

BackgroundIn the vascular system, Notch receptors and ligands are expressed mainly on arteries, with Delta-like 4 (Dll4) being the only ligand known to be expressed early during the development of arterial endothelial cells and capillaries. Dll4 null embryos die very early in development with severely reduced arterial calibre and lumen and loss of arterial cell identity.ResultsThe current detailed analysis of these mutants shows that the arterial defect precedes the initiation of blood flow and that the arterial Dll4-/- endothelial cells proliferate and migrate more actively. Dll4-/- mutants reveal a defective basement membrane around the forming aorta and increased endothelial cell migration from the dorsal aorta to peripheral regions, which constitute the main causes of arterial lumen reduction in these embryos. The increased proliferation and migration of Dll4-/- endothelial cells was found to coincide with increased expression of the receptors VEGFR-2 and Robo4 and with downregulation of the TGF-β accessory receptor Endoglin.ConclusionTogether, these results strongly suggest that Notch signalling can increase arterial stability and calibre by decreasing the response of arterial endothelial cells to local gradients of pro-angiogenic factors like VEGF.

Endothelial Jagged1 Antagonizes Dll4 Regulation of Endothelial Branching and Promotes Vascular Maturation Downstream of Dll4/Notch1

Objective— Notch signaling controls cardiovascular development and has been associated with several pathological conditions. Among its ligands, Jagged1 and Dll4 were shown to have opposing effects in developmental angiogenesis, but the underlying mechanism and the role of Jagged1/Notch signaling in adult angiogenesis remain incompletely understood. The current study addresses the importance of endothelial Jagged1-mediated Notch signaling in the context of adult physiological angiogenesis and the interactions of Jagged1 and Dll4 on angiogenic response and vascular maturation processes. Approach and Results— The role of endothelial Jagged1 in wound healing kinetics and angiogenesis was investigated with endothelial-specific Jag1 gain-of-function and loss-of-function mouse mutants (eJag1OE and eJag1cKO). To study the interactions between the 2 Notch ligands, genetic mouse models were combined with pharmacological inhibition of Dll4 or Jagged1, respectively. Jagged1 overexpression in endothelial cells increased vessel density, maturation, and perfusion, thus accelerating wound healing. The opposite effect was seen in eJag1cKO animals. Interestingly, Dll4 blockade in these animals led to an increase in vascular density but induced a greater decrease in perivascular cell coverage. However, Jagged1 inhibition in Dll4 gain-of-function (eDll4OE) mutants, with reduced angiogenesis, further diminished angiogenic growth and hampered perivascular cell coverage. Our findings suggest that as Dll4 blocks endothelial activation through Notch1 signaling, it also induces Jagged1 expression. Jagged1 then blocks Dll4 signaling through Notch1, allowing endothelial activation by vascular endothelial growth factor and endothelial layer growth. Jagged1 also initiates maturation of the newly formed vessels, possibly by binding and activating endothelial Notch4. Importantly, mice administered with a Notch4 agonistic antibody mimicked the mural cell phenotype of eJag1OE mutants without affecting angiogenic growth, which is thought to be Notch1 dependent. Conclusions— Endothelial Jagged1 is likely to operate downstream of Dll4/Notch1 signaling to activate Notch4 and regulate vascular maturation. Thus, Jagged1 not only counteracts Dll4/Notch in the endothelium but also generates a balance between angiogenic growth and maturation processes in vivo.

Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models

Arteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Delta-like 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic loss- and gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4+/- mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4+/- mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

Notch1 is pan-endothelial at the onset of flow and regulated by flow

Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (≥10 dynes/cm2). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.

Dynamics of Notch pathway expression during mouse testis post-natal development and along the spermatogenic cycle.

The transcription and expression patterns of Notch pathway components (Notch 1–3, Delta1 and 4, Jagged1) and effectors (Hes1, Hes2, Hes5 and Nrarp) were evaluated (through RT-PCR and IHC) in the mouse testis at key moments of post-natal development, and along the adult spermatogenic cycle. Notch pathway components and effectors are transcribed in the testis and expressed in germ, Sertoli and Leydig cells, and each Notch component shows a specific cell-type and time-window expression pattern. This expression at key testis developmental events prompt for a role of Notch signaling in pre-pubertal spermatogonia quiescence, onset of spermatogenesis, and regulation of the spermatogenic cycle.

Low-dosage inhibition of Dll4 signaling promotes wound healing by inducing functional neo-angiogenesis.

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing.