Peetra U. Magnusson

VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) activation by VEGF‐A is essential in vasculogenesis and angiogenesis. We have generated a pan‐phosphorylation site map of VEGFR‐2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C‐terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR‐2‐expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T‐cell‐specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF‐A‐induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd‐deficient mice, indicating a critical role of Y951‐TSAd signaling in pathological angiogenesis.

Are Therapeutic Human Mesenchymal Stromal Cells Compatible with Human Blood

Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture‐expanded human MSCs elicit an innate immune attack, termed instant blood‐mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell‐passage number. Short‐term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D‐dimer or acute‐phase reactants with the currently applied dose of 1.0–3.0 × 106 cells per kilogram. Culture‐expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose‐dependent and increases after prolonged ex vivo expansion. Currently applied doses of low‐passage clinical‐grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells. Stem Cells2012;30:1565–1574

Formation of Composite Endothelial Cell-Mesenchymal Stem Cell-Islets; a novel approach to promote islet revascularization

OBJECTIVE—Mesenchymal stem cells (MSCs) contribute to endothelial cell (EC) migration by producing proteases, thereby paving the way into the tissues for ECs. MSCs were added to our previously described composite EC islets as a potential means to improve their capacity for islet angiogenesis. RESEARCH DESIGN AND METHODS—Human islets were coated with primary human bone marrow–derived MSCs and dermal microvascular ECs. The capacity of ECs, with or without MSCs, to adhere to and grow into human islets was analyzed. The survival and functionality of these composite islets were evaluated in a dynamic perifusion assay, and their capacity for angiogenesis in vitro was assessed in a three-dimensional fibrin gel assay. RESULTS—ECs proliferated after culture in MSC-conditioned medium, and MSCs improved the EC coverage threefold compared with EC islets alone. Islet survival in vitro and the functionality of the composite islets after culture were equal to those of control islets. The EC-MSC islets showed a twofold increase in total sprout formation compared with EC islets, and vascular sprouts emanating from the EC-MSC–islet surface showed migration of ECs into the islets and also into the surrounding matrix, either alone or in concert with MSCs. CONCLUSIONS—EC proliferation, sprout formation, and ingrowth of ECs into the islets were enhanced by MSCs. The use of composite EC-MSC islets may have beneficial effects on revascularization and immune regulation. The technique presented allows for pretreatment of donor islets with recipient-derived ECs and MSCs as a means of improving islet engraftment.

Angiostatin and endostatin inhibit endothelial cell migration in response to FGF and VEGF without interfering with specific intracellular signal transduction pathways.

The anti‐angiogenic agents angiostatin and endostatin have been shown to affect endothelial cell migration in a number of studies. We have examined the effect of these agents on intracellular signalling pathways known to regulate endothelial cell migration and proliferation/survival. Both agents inhibited fibroblast growth factor (FGF)‐, and vascular endothelial growth factor (VEGF)‐mediated migration of primary human microvascular endothelial cells and affected vascular formation in the embryoid body model. However, using phosphospecific antibodies we could not detect any effect of angiostatin or endostatin on phospholipase C‐γ (PLC‐γ), Akt/PKB, p44/42 mitogen‐activated protein kinase (MAPK), p38 MAPK and p21‐activated kinase (PAK) activity. Furthermore, using a glutathione S‐transferase (GST)‐PAK pull‐down assay, we could not detect any effect on Rac activity. We conclude that angiostatin and endostatin inhibit chemotaxis, without affecting intracellular signalling pathways known to regulate endothelial migration and proliferation/survival.

CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model.MethodsCD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.ResultsThe engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.ConclusionCNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

Deregulation of Flk-1/vascular endothelial growth factor receptor-2 in fibroblast growth factor receptor-1-deficient vascular stem cell development.

We have employed embryoid bodies derived from murine embryonal stem cells to study effects on vascular development induced by fibroblast growth factor (FGF)-2 and FGF receptor-1, in comparison to the established angiogenic factor vascular endothelial growth factor (VEGF)-A and its receptor VEGF receptor-2. Exogenous FGF-2 promoted formation of morphologically distinct, long slender vessels in the embryoid bodies, whereas VEGF-A-treated bodies displayed a compact plexus of capillaries. FGF-2 stimulation of embryonal stem cells under conditions where VEGF-A/VEGFR-2 function was blocked, led to formation of endothelial cell clusters, which failed to develop into vessels. FGFR-1-/- embryoid bodies responded to VEGF-A by establishment of the characteristic vascular plexus, but FGF-2 had no effect on vascular development in the absence of FGFR-1. The FGFR-1-/- embryoid bodies displayed considerably increased basal level of vessel formation, detected by immunohistochemical staining for platelet-endothelial cell adhesion molecule (PECAM)/CD31. This basal vascularization was blocked by neutralizing antibodies against VEGFR-2 or VEGF-A and biochemical analyses indicated changes in regulation of VEGFR-2 in the absence of FGFR-1 expression. We conclude that VEGF-A/VEGFR-2-dependent vessel formation occurs in the absence of FGF-2/FGFR-1, which, however, serve to modulate vascular development.

Platelet-Derived Growth Factor Receptor-β Constitutive Activity Promotes Angiogenesis In Vivo and In Vitro

Objective— Knockout studies have demonstrated crucial roles for the platelet-derived growth factor-B and its cognate receptor, platelet-derived growth factor receptor-&bgr; (PDGFR-&bgr;), in blood vessel maturation, that is, the coverage of newly formed vessels with mural cells/pericytes. This study describes the consequences of a constitutively activating mutation of the PDGFR-&bgr; (PdgfrbD849V) introduced into embryonic stem cells with respect to vasculogenesis/angiogenesis in vitro and in vivo. Methods and Results— Embryonic stem cells were induced to either form teratomas in vivo or embryoid bodies, an in vitro model for mouse embryogenesis. Western blotting studies on embryoid bodies showed that expression of a single allele of the mutant Pdgfrb led to increased levels of PDGFR-&bgr; tyrosine phosphorylation and augmented downstream signal transduction. This was accompanied by enhanced vascular development, followed by exaggerated angiogenic sprouting with abundant pericyte coating as shown by immunohistochemistry/immunofluorescence. PdgfrbD849V/+ embryoid bodies were characterized by increased expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor-2; neutralizing antibodies against VEGF-A/VEGF receptor-2 blocked vasculogenesis and angiogenesis in mutant embryoid bodies. Moreover, PdgfrbD849V/+ embryonic stem cell–derived teratomas in nude mice were more densely vascularized than wild-type teratomas. Conclusion— Increased PDGFR-&bgr; kinase activity is associated with elevated expression of VEGF-A and VEGF receptor-2, acting directly on endothelial cells and resulting in increased vessel formation.

Anchoring of Vascular Endothelial Growth Factor to Surface-Immobilized Heparin on Pancreatic Islets: Implications for Stimulating Islet Angiogenesis

In pancreatic islet transplantation, early revascularization is necessary for long-term graft function. We have shown in in vitro and in vivo models that modification with surface-attached heparin protects the islets from acute attack by the innate immune system of the blood following intraportal islet transplantation. In this study, we have investigated the ability of an immobilized conjugate composed of heparin to bind the angiogenic growth factor vascular endothelial growth factor-A (VEGF-A) as a means of attracting endothelial cells (ECs) to induce angiogenesis and revascularization. We analyzed the capacity of VEGF-A to bind to immobilized heparin and how this affected the proliferation and adherence of ECs to both artificial glass surfaces and islets. Quartz crystal microbalance with dissipation monitoring and slot-blot demonstrated the binding of VEGF-A to heparin-coated surfaces upon which ECs showed protein-dependent proliferation. Also, ECs cultured on heparin-coated glass surfaces exhibited effects upon focal contacts. Heparinized islets combined with VEGF-A demonstrated unaffected insulin release. Further, covering islets with heparin also increased the adhesion of ECs to the islet surface. Immobilized heparin on the islet surface may be a useful anchor molecule for achieving complete coverage of islets with angiogenic growth factors, ultimately improving islet revascularization and engraftment in pancreatic islet transplantation.

Autoregulation of thromboinflammation on biomaterial surfaces by a multicomponent therapeutic coating

Abstract Activation of the thrombotic and complement systems is the main recognition and effector mechanisms in the multiple adverse biological responses triggered when biomaterials or therapeutic cells come into blood contact. We have created a surface which is auto-protective to human innate immunity by combining three fundamentally different strategies, all developed by us previously, which have been shown to induce substantial, but incomplete hemocompatibility when used separately. In summary, we have conjugated a factor H–binding peptide; and an ADP-degrading enzyme; using a PEG linker on both material and cellular surfaces. When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated, by degrading ADP. Thus, by inhibiting thromboinflammation using a multicomponent approach, we have created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation.

Fibroblast Growth Factor Receptor-1 Expression Is Required for Hematopoietic but not Endothelial Cell Development

Objective—The purpose of this study was to clarify the role of fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in hematopoietic/endothelial development. Methods and Results—Using several different FGFR-1–specific antibodies and FGFR-1 promoter-driven LacZ activity, we show that FGFR-1 is expressed and active as a tyrosine kinase in a subpopulation of endothelial cells (≈20% of the endothelial pool) during development in embryoid bodies. In agreement, in stem cell-derived teratomas, expression of FGFR-1 was detected in some but not all vessels. The FGFR-1 expressing endothelial cells were mitogenically active in the absence and presence of vascular endothelial growth factor (VEGF). Expression of FGFR-1 in endothelial cell precursors was not required for vascular development, and vascularization was enhanced in FGFR-1–deficient embryoid bodies compared with wild-type stem cells. In contrast, hematopoietic development was severely disturbed, with reduced expression of markers for primitive and definitive hematopoiesis. Conclusions—Our data show that FGFR-1 is expressed in early hematopoietic/endothelial precursor cells, as well as in a subpool of endothelial cells in tumor vessels, and that it is critical for hematopoietic but not for vascular development.