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https://orcid.org/0000-0003-1898-4928All Publications
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A human arteriovenous differentiation roadmap reveals vein developmental mechanisms and vascular effects of viruses.
bioRxiv : the preprint server for biology
2025
Abstract
We map human artery and vein endothelial cell (EC) differentiation from pluripotent stem cells, and employ this roadmap to discover new mechanisms of vascular development (vein differentiation) and disease (viral infection). We discovered vein development unfolds in two steps driven by opposing signals: VEGF differentiates mesoderm into "pre-vein" ECs, but surprisingly, VEGF/ERK inhibition subsequently specifies vein ECs. Pre-vein ECs co-expressed certain arterial (SOX17) and venous (APLNR) markers, harbored poised chromatin at future venous genes, but completed venous differentiation only upon VEGF inhibition. Intersectional lineage tracing revealed that early Sox17+ Aplnr+ ECs also formed veins in vivo. Next, we compared how Ebola, Andes, and Nipah viruses infect artery and vein ECs under biosafety-level-4 containment. Each virus distinctly affected ECs. Interestingly, artery and vein ECs also responded divergently to the same virus, thus revealing that developmentally-specified cell identity impacts viral infection. Collectively, this arteriovenous differentiation roadmap illuminates vascular development and disease.
View details for DOI 10.1101/2025.10.11.681838
View details for PubMedID 41279810
View details for PubMedCentralID PMC12632389
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Fetal-like reversion in the regenerating intestine is regulated by mesenchymal asporin.
Cell stem cell
2025
Abstract
Mesenchymal cells and the extracellular matrix (ECM) support epithelium during homeostasis and regeneration. However, the role of the mesenchyme in epithelial conversion into a fetal-like regenerative state after damage is not known. We modeled epithelial regeneration by culturing intestinal epithelium on decellularized small intestinal scaffolds (iECM) and identify asporin (Aspn), an ECM-bound proteoglycan, as a critical mediator of epithelial fetal-like reprogramming. After damage, transient increase in Aspn expression by the pericryptal fibroblasts induces epithelial transforming growth factor β (TGF-β)-signaling via CD44 and promotes timely epithelial reprogramming. Temporal control of Aspn is lost in old mice, and after damage, the persistently high level of Aspn stagnates epithelium in the regenerative state. Increase in Wnt signaling can resolve the stagnated regenerative program of the old epithelium, promoting restoration of tissue function. In summary, we establish a platform for modeling epithelial injury responses ex vivo and show that the mesenchymal Aspn-producing niche modulates tissue repair by regulating epithelial fetal-like reprogramming.
View details for DOI 10.1016/j.stem.2025.02.009
View details for PubMedID 40054463
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Protocol for efficient generation of human artery and vein endothelial cells from pluripotent stem cells.
STAR protocols
2024; 6 (1): 103494
Abstract
Blood vessels permeate all organs and execute myriad roles in health and disease. Here, we present a protocol to efficiently generate human artery and vein endothelial cells (ECs) from pluripotent stem cells within 3-4 days of differentiation. We delineate how to seed human pluripotent stem cells and sequentially differentiate them into primitive streak, lateral mesoderm, and either artery or vein ECs. We differentiate stem cells in defined, serum-free culture media in monolayers, without feeder cells or genetic manipulations. For complete details on the use and execution of this protocol, please refer to Ang et al. 1.
View details for DOI 10.1016/j.xpro.2024.103494
View details for PubMedID 39705146
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Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression
JOURNAL OF CLINICAL INVESTIGATION
2022; 132 (15)
Abstract
Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C-induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C-induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.
View details for DOI 10.1172/JCI155478
View details for Web of Science ID 000842455700004
View details for PubMedID 35763346
View details for PubMedCentralID PMC9337826
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Blood flow modeling reveals improved collateral artery performance during the regenerative period in mammalian hearts.
Nature cardiovascular research
2022; 1 (8): 775-790
Abstract
Collateral arteries bridge opposing artery branches, forming a natural bypass that can deliver blood flow downstream of an occlusion. Inducing coronary collateral arteries could treat cardiac ischemia, but more knowledge on their developmental mechanisms and functional capabilities is required. Here we used whole-organ imaging and three-dimensional computational fluid dynamics modeling to define spatial architecture and predict blood flow through collaterals in neonate and adult mouse hearts. Neonate collaterals were more numerous, larger in diameter and more effective at restoring blood flow. Decreased blood flow restoration in adults arose because during postnatal growth coronary arteries expanded by adding branches rather than increasing diameters, altering pressure distributions. In humans, adult hearts with total coronary occlusions averaged 2 large collaterals, with predicted moderate function, while normal fetal hearts showed over 40 collaterals, likely too small to be functionally relevant. Thus, we quantify the functional impact of collateral arteries during heart regeneration and repair-a critical step toward realizing their therapeutic potential.
View details for DOI 10.1038/s44161-022-00114-9
View details for PubMedID 37305211
View details for PubMedCentralID PMC10256232
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Common Laboratory Mice Are Susceptible to Infection with the SARS-CoV-2 Beta Variant
VIRUSES-BASEL
2021; 13 (11)
Abstract
Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern. This suggests that these variants might have the ability to infect common laboratory mice. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes within 2-3 days post infection, consistent with results seen in other murine models of COVID-19, at a reasonable virus dose (2 × 105 PFU). The findings suggest that common laboratory mice can serve as the animal model of choice for testing the effectiveness of antiviral drugs and vaccines against SARS-CoV-2.
View details for DOI 10.3390/v13112263
View details for Web of Science ID 000729154300001
View details for PubMedID 34835069
View details for PubMedCentralID PMC8619350
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Characterization of <i>ANGPT2</i> mutations associated with primary lymphedema
SCIENCE TRANSLATIONAL MEDICINE
2020; 12 (560)
Abstract
Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) receptor complex and α5β1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)-ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.
View details for DOI 10.1126/scitranslmed.aax8013
View details for Web of Science ID 000567648900003
View details for PubMedID 32908006
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Investigation on the role of biallelic variants in <i>VEGF-C</i> found in a patient affected by Milroy-like lymphedema
MOLECULAR GENETICS & GENOMIC MEDICINE
2020; 8 (9): e1389
Abstract
Milroy-like disease is the diagnostic definition used for patients with phenotypes that resemble classic Milroy disease (MD) but are negative to genetic testing for FLT4. In this study, we aimed at performing a genetic characterization and biochemical analysis of VEGF-C variations found in a female proband born with congenital edema consistent with Milroy-like disease.The proband underwent next-generation sequencing-based genetic testing for a panel of genes associated with known forms of hereditary lymphedema. Segregation analysis was performed on family members by direct sequencing. In vitro studies were performed to evaluate the role of a novel identified variant.Two VEGF-C variations were found in the proband, a novel p.(Ser65Arg) and a pathogenic c.148-3_148-2delCA, of paternal and maternal origin, respectively. Functional characterization of the p.(Ser65Arg) variation in vitro showed alterations in VEGF-C processing.Our findings reveal an interesting case in which biallelic variants in VEGF-C are found in a patient with Milroy-like lymphedema. These data expand our understanding of the etiology of congenital Milroy-like lymphedema.
View details for DOI 10.1002/mgg3.1389
View details for Web of Science ID 000543340000001
View details for PubMedID 32592340
View details for PubMedCentralID PMC7507552
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KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D
ELIFE
2019; 8
Abstract
Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, for example in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D.
View details for DOI 10.7554/eLife.44478
View details for Web of Science ID 000472554800001
View details for PubMedID 31099754
View details for PubMedCentralID PMC6588350
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Biology of Vascular Endothelial Growth Factor C in the Morphogenesis of Lymphatic Vessels
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
2018; 6: 7
Abstract
Because virtually all tissues contain blood vessels, the importance of hemevascularization has been long recognized in regenerative medicine and tissue engineering. However, the lymphatic vasculature has only recently become a subject of interest. Central to the task of growing a lymphatic network are lymphatic endothelial cells (LECs), which constitute the innermost layer of all lymphatic vessels. The central molecule that directs proliferation and migration of LECs during embryogenesis is vascular endothelial growth factor C (VEGF-C). VEGF-C is therefore an important ingredient for LEC culture and attempts to (re)generate lymphatic vessels and networks. During its biosynthesis VEGF-C undergoes a stepwise proteolytic processing, during which its properties and affinities for its interaction partners change. Many of these fundamental aspects of VEGF-C biosynthesis have only recently been uncovered. So far, most-if not all-applications of VEGF-C do not discriminate between different forms of VEGF-C. However, for lymphatic regeneration and engineering purposes, it appears mandatory to understand these differences, since they relate, e.g., to important aspects such as biodistribution and receptor activation potential. In this review, we discuss the molecular biology of VEGF-C as it relates to the growth of LECs and lymphatic vessels. However, the properties of VEGF-C are similarly relevant for the cardiovascular system, since both old and recent data show that VEGF-C can have a profound effect on the blood vasculature.
View details for DOI 10.3389/fbioe.2018.00007
View details for Web of Science ID 000440252700003
View details for PubMedID 29484295
View details for PubMedCentralID PMC5816233
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Key molecules in lymphatic development, function, and identification
ANNALS OF ANATOMY-ANATOMISCHER ANZEIGER
2018; 219: 25-34
Abstract
While both blood and lymphatic vessels transport fluids and thus share many similarities, they also show functional and structural differences, which can be used to differentiate them. Specific visualization of lymphatic vessels has historically been and still is a pivot point in lymphatic research. Many of the proteins that are investigated by molecular biologists in lymphatic research have been defined as marker molecules, i.e. to visualize and distinguish lymphatic endothelial cells (LECs) from other cell types, most notably from blood vascular endothelial cells (BECs) and cells of the hematopoietic lineage. Among the factors that drive the developmental differentiation of lymphatic structures from venous endothelium, Prospero homeobox protein 1 (PROX1) is the master transcriptional regulator. PROX1 maintains lymphatic identity also in the adult organism and thus is a universal LEC marker. Vascular endothelial growth factor receptor-3 (VEGFR-3) is the major tyrosine kinase receptor that drives LEC proliferation and migration. The major activator for VEGFR-3 is vascular endothelial growth factor-C (VEGF-C). However, before VEGF-C can signal, it needs to be proteolytically activated by an extracellular protein complex comprised of Collagen and calcium binding EGF domains 1 (CCBE1) protein and the protease A disintegrin and metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). This minireview attempts to give an overview of these and a few other central proteins that scientific inquiry has linked specifically to the lymphatic vasculature. It is limited in scope to a brief description of their main functions, properties and developmental roles.
View details for DOI 10.1016/j.aanat.2018.05.003
View details for Web of Science ID 000443531900003
View details for PubMedID 29842991
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Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1
SCIENTIFIC REPORTS
2017; 7: 4916
Abstract
The collagen- and calcium-binding EGF domains 1 (CCBE1) protein is necessary for lymphangiogenesis. Its C-terminal collagen-like domain was shown to be required for the activation of the major lymphangiogenic growth factor VEGF-C (Vascular Endothelial Growth Factor-C) along with the ADAMTS3 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-3) protease. However, it remained unclear how the N-terminal domain of CCBE1 contributed to lymphangiogenic signaling. Here, we show that efficient activation of VEGF-C requires its C-terminal domain both in vitro and in a transgenic mouse model. The N-terminal EGF-like domain of CCBE1 increased VEGFR-3 signaling by colocalizing pro-VEGF-C with its activating protease to the lymphatic endothelial cell surface. When the ADAMTS3 amounts were limited, proteolytic activation of pro-VEGF-C was supported by the N-terminal domain of CCBE1, but not by its C-terminal domain. A single amino acid substitution in ADAMTS3, identified from a lymphedema patient, was associated with abnormal CCBE1 localization. These results show that CCBE1 promotes VEGFR-3 signaling and lymphangiogenesis by different mechanisms, which are mediated independently by the two domains of CCBE1: by enhancing the cleavage activity of ADAMTS3 and by facilitating the colocalization of VEGF-C and ADAMTS3. These new insights should be valuable in developing new strategies to therapeutically target VEGF-C/VEGFR-3-induced lymphangiogenesis.
View details for DOI 10.1038/s41598-017-04982-1
View details for Web of Science ID 000404970900078
View details for PubMedID 28687807
View details for PubMedCentralID PMC5501841
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Factors regulating the substrate specificity of cytosolic phospholipase A<sub>2</sub>-alpha in vitro
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS
2016; 1861 (11): 1597-1604
Abstract
Cytosolic phospholipase A2 alpha (cPLA2α) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA2α for AA-containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA2α towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the sn1 or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA2α. Such promiscuous binding would explain the previous finding that cPLA2α has both PLA1 and PLA2 activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA2α is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA2α.
View details for DOI 10.1016/j.bbalip.2016.06.022
View details for Web of Science ID 000385325800001
View details for PubMedID 27377346
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Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells
MOLECULAR CANCER
2016; 15: 34
Abstract
Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive type of cancer that lacks effective targeted therapy. Despite detailed molecular profiling, no targeted therapy has been established. Hence, with the aim of gaining deeper understanding of the functional differences of TNBC subtypes and how that may relate to potential novel therapeutic strategies, we studied comprehensive anticancer-agent responses among a panel of TNBC cell lines.The responses of 301 approved and investigational oncology compounds were measured in 16 TNBC cell lines applying a functional profiling approach. To go beyond the standard drug viability effect profiling, which has been used in most chemosensitivity studies, we utilized a multiplexed readout for both cell viability and cytotoxicity, allowing us to differentiate between cytostatic and cytotoxic responses.Our approach revealed that most single-agent anti-cancer compounds that showed activity for the viability readout had no or little cytotoxic effects. Major compound classes that exhibited this type of response included anti-mitotics, mTOR, CDK, and metabolic inhibitors, as well as many agents selectively inhibiting oncogene-activated pathways. However, within the broad viability-acting classes of compounds, there were often subsets of cell lines that responded by cell death, suggesting that these cells are particularly vulnerable to the tested substance. In those cases we could identify differential levels of protein markers associated with cytotoxic responses. For example, PAI-1, MAPK phosphatase and Notch-3 levels associated with cytotoxic responses to mitotic and proteasome inhibitors, suggesting that these might serve as markers of response also in clinical settings. Furthermore, the cytotoxicity readout highlighted selective synergistic and synthetic lethal drug combinations that were missed by the cell viability readouts. For instance, the MEK inhibitor trametinib synergized with PARP inhibitors. Similarly, combination of two non-cytotoxic compounds, the rapamycin analog everolimus and an ATP-competitive mTOR inhibitor dactolisib, showed synthetic lethality in several mTOR-addicted cell lines.Taken together, by studying the combination of cytotoxic and cytostatic drug responses, we identified a deeper spectrum of cellular responses both to single agents and combinations that may be highly relevant for identifying precision medicine approaches in TNBC as well as in other types of cancers.
View details for DOI 10.1186/s12943-016-0517-3
View details for Web of Science ID 000376146000002
View details for PubMedID 27165605
View details for PubMedCentralID PMC4862054