Elisabet Wallgard

Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis

In sprouting angiogenesis, specialized endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A. VEGF-A is also essential for the induction of endothelial tip cells, but it is not known how single tip cells are selected to lead each vessel sprout, and how tip-cell numbers are determined. Here we present evidence that delta-like 4 (Dll4)–Notch1 signalling regulates the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the mouse retina. We show that inhibition of Notch signalling using γ-secretase inhibitors, genetic inactivation of one allele of the endothelial Notch ligand Dll4, or endothelial-specific genetic deletion of Notch1, all promote increased numbers of tip cells. Conversely, activation of Notch by a soluble jagged1 peptide leads to fewer tip cells and vessel branches. Dll4 and reporters of Notch signalling are distributed in a mosaic pattern among endothelial cells of actively sprouting retinal vessels. At this location, Notch1-deleted endothelial cells preferentially assume tip-cell characteristics. Together, our results suggest that Dll4–Notch1 signalling between the endothelial cells within the angiogenic sprout serves to restrict tip-cell formation in response to VEGF, thereby establishing the adequate ratio between tip and stalk cells required for correct sprouting and branching patterns. This model offers an explanation for the dose-dependency and haploinsufficiency of the Dll4 gene, and indicates that modulators of Dll4 or Notch signalling, such as γ-secretase inhibitors developed for Alzheimer’s disease, might find usage as pharmacological regulators of angiogenesis.

Pericytes regulate the blood–brain barrier

The blood–brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB.

Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.

Large-scale identification of genes implicated in kidney glomerulus development and function

To advance our understanding of development, function and diseases in the kidney glomerulus, we have established and large‐scale sequenced cDNA libraries from mouse glomeruli at different stages of development, resulting in a catalogue of 6053 different genes. The glomerular cDNA clones were arrayed and hybridized against a series of labeled targets from isolated glomeruli, non‐glomerular kidney tissue, FACS‐sorted podocytes and brain capillaries, which identified over 300 glomerular cell‐enriched transcripts, some of which were further sublocalized to podocytes, mesangial cells and juxtaglomerular cells by in situ hybridization. For the earliest podocyte marker identified, Foxc2, knockout mice were used to analyze the role of this protein during glomerular development. We show that Foxc2 controls the expression of a distinct set of podocyte genes involved in podocyte differentiation and glomerular basement membrane maturation. The primary podocyte defects also cause abnormal differentiation and organization of the glomerular vascular cells. We surmise that studies on the other novel glomerulus‐enriched transcripts identified in this study will provide new insight into glomerular development and pathomechanisms of disease.

Identification of a Core Set of 58 Gene Transcripts With Broad and Specific Expression in the Microvasculature

Objective—Pathological angiogenesis is an integral component of many diseases. Antiangiogenesis and vascular targeting are therefore promising new therapeutic principles. However, few endothelial-specific putative drug targets have been identified, and information is still limited about endothelial-specific molecular processes. Here we aimed at determining the endothelial cell-specific core transcriptome in vivo. Methods and Results—Analysis of publicly available microarray data identified a mixed vascular/lung cluster of 132 genes that correlated with known endothelial markers. Filtering against kidney glomerular/nonglomerular and brain vascular/nonvascular microarray profiles separated contaminating lung markers, leaving 58 genes with broad and specific microvascular expression. More than half of these have not previously been linked to endothelial functions or studied in detail before. The endothelial cell-specific expression of a selected subset of these, Eltd1, Gpr116, Ramp2, Slc9a3r2, Slc43a3, Rasip1, and NM_023516, was confirmed by real-time quantitative polymerase chain reaction and/or immunohistochemistry. Conclusions—We have used a combination of publicly available and own microarray data to identify 58 gene transcripts with broad yet specific expression in microvascular endothelium. Most of these have unknown functions, but many of them are predicted to be cell surface expressed or implicated in cell signaling processes and should therefore be explored as putative microvascular drug targets.

Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma

The Notch ligand delta-like ligand 4 (DLL4) is an essential component expressed by endothelial tip cells during angiogenic sprouting. We have described a conceptually novel therapeutic strategy for targeting tumor angiogenesis and endothelial tip cells based on DNA vaccination against DLL4. Immunization with DLL4-encoding plasmid DNA by in vivo electroporation severely retarded the growth of orthotopically implanted mammary carcinomas in mice by induction of a nonproductive angiogenic response. Mechanistically, vaccination brought about a break in tolerance against the self-antigen, DLL4, as evidenced by the production of inhibitory and inherently therapeutic antibodies against mouse DLL4. Importantly, no evidence for a delayed wound healing response, or for toxicity associated with pharmacological blockade of DLL4 signaling, was noted in mice immunized with the DLL4 vaccine. We have thus developed a well-tolerated DNA vaccination strategy targeting the endothelial tip cells and the antigen DLL4 with proven therapeutic efficacy in mouse models of mammary carcinoma; a disease that has been reported to dramatically induce the expression of DLL4. Conceivably, induction of immunity toward principal mediators of pathological angiogenesis could provide protection against recurrent malignant disease in the adjuvant setting.

Combination of reverse and chemical genetic screens reveals angiogenesis inhibitors and targets.

We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development.

Background: Angiogenesis is implicated in many pathological conditions. The role of the proteins involved remains largely unknown, and few vascular‐specific drug targets have been discovered. Previously, in a screen for angiogenesis regulators, we identified Paladin (mouse: X99384, human: KIAA1274), a protein containing predicted S/T/Y phosphatase domains. Results: We present a mouse knockout allele for Paladin with a β‐galactosidase reporter, which in combination with Paladin antibodies demonstrate that Paladin is expressed in the vasculature. During mouse embryogenesis, Paladin is primarily expressed in capillary and venous endothelial cells. In adult mice Paladin is predominantly expressed in arterial pericytes and vascular smooth muscle cells. Paladin also displays vascular‐restricted expression in human brain, astrocytomas, and glioblastomas. Conclusions: Paladin, a novel putative phosphatase, displays a dynamic expression pattern in the vasculature. During embryonic stages it is broadly expressed in endothelial cells, while in the adult it is selectively expressed in arterial smooth muscle cells. Developmental Dynamics 241:770–786, 2012.