Biomedical subjects

Lena Claesson-Welsh

Publications and source records attributed to Lena Claesson-Welsh.

At least 19 recordsLinked to original sources

PubMed2006

Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions.

Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.

Actins↗

PubMed2006

Platelet-derived growth factor receptor-beta promotes early endothelial cell differentiation.

Platelet-derived growth factor BB (PDGF-BB) has been assigned a critical role in vascular stability by promoting the recruitment of PDGF receptor-beta-expressing perivascular cells. Here we present data indicating that early hematopoietic/endothelial (hemangio) precursors express PDGFR-beta based on coexpression with CD31, vascular endothelial growth factor receptor-2, and CD41 in 2 models: mouse yolk sac (embryonic day 8 [E8]) and differentiating mouse embryonic stem cells (embryoid bodies). Expression of PDGFR-beta on hemangioprecursor cells in the embryoid bodies gradually disappeared, and, at E14, expression appeared on perivascular cells. Activation of the PDGFR-beta on the hemangioprecursors accelerated the differentiation of endothelial cells, whereas differentiation of the hematopoietic lineage was suppressed. In E9.5 yolk sacs derived from recombinant mice expressing kinase-active PDGFR-beta with an aspartic acid to asparagine (D894N) replacement in the kinase activating loop and from mice with ubiquitous expression of PDGF-BB driven by the Rosa26 locus, the number of CD41-expressing early hematopoietic cells decreased by 36% and 34%, respectively, compared with staged wild-type littermates. Moreover, enhanced vascular remodeling was evident in the Rosa26-PDGF-BB yolk sacs. We conclude that PDGFR-beta is expressed on early hemangioprecursor cells, regulating vascular/hematopoietic development.

Amino Acid Substitution↗

PubMed2006

Early lymph vessel development from embryonic stem cells.

OBJECTIVE: The purpose of this study was to establish a model system for lymph vessel development based on directed differentiation of murine embryonic stem cells. METHODS AND RESULTS: Stem cells were aggregated to form embryoid bodies, and subsequently cultured in 3-dimensional collagen matrix for up to 18 days. Treatment with vascular endothelial growth factor (VEGF)-C and VEGF-A individually enhanced formation of lymphatic vessel structures, although combined treatment with VEGF-C and VEGF-A was most potent and gave rise to a network of LYVE-1, podoplanin, Prox1, and VEGF receptor-3 positive lymphatic vessel structures running parallel to and apparently emanating from, capillaries. In contrast, fibroblast growth factor-2, hepatocyte growth factor, or hypoxia had little or no effect on the development of the early lymphatics. Further, cells of hematopoietic origin were shown to express lymphatic markers. In summary, different subpopulations of lymphatic endothelial cells were identified on the basis of differential expression of several lymphatic and blood vessel markers, indicating vascular heterogeneity. CONCLUSIONS: We conclude that the present model closely mimics the early steps of lymph vessel development in mouse embryos.

Animals↗

PubMed2006

Minimal active domain and mechanism of action of the angiogenesis inhibitor histidine-rich glycoprotein.

Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein that efficiently arrests growth and vascularization of mouse tumor models. We have shown that the antiangiogenic effect of HRGP is dependent on its histidine/proline-rich domain, which needs to be released from the mother protein to exert its effects. Here we identify a 35-amino-acid peptide, HRGP330, derived from the histidine/proline-rich domain as endowed with antiangiogenic properties in vitro and in vivo. The mechanism of action of HRGP330 involves subversion of focal adhesion function by disruption of integrin-linked kinase (ILK) and focal adhesion kinase (FAK) functions, inhibition of vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of the FAK substrate alpha-actinin, and, as a consequence, an arrest in endothelial cell motility. The disturbed focal adhesion function is reflected in the ability of HRGP as well as of HRGP330 to prevent endothelial cell adhesion to vitronectin in a manner involving alpha(v)beta3 integrin. In conclusion, HRGP330, which we define as the minimal antiangiogenic domain of HRGP, exerts its effects through signal transduction targeting focal adhesions, thereby interrupting VEGF-induced endothelial cell motility.

Actinin↗

PubMed2006

The anti-angiogenic His/Pro-rich fragment of histidine-rich glycoprotein binds to endothelial cell heparan sulfate in a Zn2+-dependent manner.

The plasma protein histidine-rich glycoprotein (HRGP), which has been identified as an angiogenesis inhibitor, binds to heparan sulfate (HS) in a Zn(2+)-dependent manner. We wished to test whether this interaction is mechanistically important in mediation of the anti-angiogenic effect of HRGP. Inhibition of angiogenesis by HRGP is exerted through its central His/Pro-rich domain, which is proteolytically released. A 35-amino-acid residue synthetic peptide, HRGP330, derived from the His/Pro-rich domain retains the inhibitory effect on blood vessel formation in vitro and in vivo, an effect dependent on the presence of Zn(2+). We now show that HRGP330 binds heparin/HS with the same capacity as full-length HRGP, and the binding is Zn(2+)-dependent. Peptides derived from the His/Pro-rich domain of HRGP downstream of HRGP330 fail to inhibit endothelial cell migration and display a significantly reduced heparin-binding capacity. An even shorter peptide, HRGP335, covering a 26-amino-acid sequence within HRGP330 retains full heparin/HS-binding capacity. Characterization of the HS interaction shows that there is a tissue-specific HS pattern recognized by HRGP335 and that the minimal length of heparin/HS required for binding to HRGP335 is an 8-mer oligosaccharide. Saturation of the HS binding sites in HRGP330 by pre-incubation with heparin abrogates the HRGP330-induced rearrangement of endothelial cell focal adhesions, suggesting that interaction with cell surface HS is needed for HRGP330 to exert its anti-angiogenic effect.

Animals↗

PubMed2006

Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis.

Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.

Amidohydrolases↗

PubMed2006

VEGF receptor signalling - in control of vascular function.

Vascular endothelial growth-factor receptors (VEGFRs) regulate the cardiovascular system. VEGFR1 is required for the recruitment of haematopoietic precursors and migration of monocytes and macrophages, whereas VEGFR2 and VEGFR3 are essential for the functions of vascular endothelial and lymphendothelial cells, respectively. Recent insights have shed light onto VEGFR signal transduction and the interplay between different VEGFRs and VEGF co-receptors in development, adult physiology and disease.

Animals↗

PubMed2005

Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis.

The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.

Aging↗

PubMed2005

Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for chloride intracellular channel 4 (CLIC4) in tubular morphogenesis.

Formation of new vessels from pre-existing capillaries demands extensive reprogramming of endothelial cells through transcriptional and post-transcriptional events. We show that 120 protein spots in a two-dimensional isoelectric focusing/electrophoretic analysis were affected during vascular endothelial growth factor-A-induced endothelial cell tubular morphogenesis in vitro, as a result of changes in charge or expression level of the corresponding proteins. For about 22% of the spots, the protein products could be identified, of which several previously have been implicated in cytoskeletal reorganization and angiogenesis. One such protein was heat shock protein 27, a chaperone involved in beta-actin rearrangement that was identified as regulated in degree of serine phosphorylation. We also identified regulation of chloride intracellular channel 4 (CLIC4), the expression of which decreased during tubular morphogenesis. CLIC4 was expressed at high levels in resting vessels, whereas expression was modulated during pathological angiogenesis such as in tumor vessels. The subcellular localization of CLIC4 in endothelial cells was dependent on whether cells were engaged in proliferation or tube formation. Antisense- and small interfering RNA-mediated suppression of CLIC4 expression led to arrest in tubular morphogenesis. Our data implicate CLIC4 in formation of a vessel lumen.

Base Sequence↗

PubMed2005

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.

Actins↗

PubMed2005

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 (approximately 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.

Animals↗

PubMed2004

The minimal active domain of endostatin is a heparin-binding motif that mediates inhibition of tumor vascularization.

Endostatin constitutes the COOH-terminal 20,000 Da proteolytic fragment of collagen XVIII and has been shown to possess antiangiogenic and antitumorigenic properties. In the present study, we have investigated the role of the heparin-binding sites in the in vivo mechanism of action of endostatin. The majority of the heparin binding is mediated by arginines 155/158/184/270 in endostatin, but there is also a minor site constituted by arginines 193/194. Using endostatin mutants lacking either of these two sites, we show that inhibition of fibroblast growth factor-2-induced angiogenesis in the chicken chorioallantoic membrane requires both heparin-binding sites. In contrast, inhibition of vascular endothelial growth factor-A-induced chorioallantoic membrane angiogenesis by endostatin was only dependent on the minor heparin-binding site (R193/194). These arginines were also required for endostatin to inhibit fibroblast growth factor-2- and vascular endothelial growth factor-A-induced chemotaxis of primary endothelial cells. Moreover, we show that a synthetic peptide corresponding to amino acids 180-199 of human endostatin (which covers the minor heparin-binding site) inhibits endothelial cell chemotaxis and reduces tumor vascularization in vivo. Substitution of arginine residues 193/194 for alanine attenuates the antiangiogenic effects of the peptide. These data show an essential role for heparin binding in the antiangiogenic action of endostatin.

Amino Acid Sequence↗

PubMed2004

Laminin-1 promotes angiogenesis in synergy with fibroblast growth factor by distinct regulation of the gene and protein expression profile in endothelial cells.

Laminins are widely distributed extracellular matrix proteins. Certain laminin isoforms are predominant in vascular basement membranes and may be critical in maintaining the stability of the mature vessel. On the other hand, formation of new vessels during angiogenesis requires degradation of the basement membrane, exposing the endothelial cells to other laminin isoforms in the surrounding extracellular matrix. We studied the effects of laminin-1 (LN-1) in different in vitro and in vivo models for angiogenesis. LN-1 induced angiogenesis in the chicken chorioallantoic membrane to the same extent as fibroblast growth factor-2 (FGF-2), and vascular development in embryoid bodies was stimulated in a synergistic manner by FGF-2 and LN-1. LN-1 promoted differentiation of endothelial cells in three-dimensional collagen gels, both in the absence and presence of FGF-2. Formation of tubular structures induced by LN-1 was accompanied by increased expression of Jagged-1, a marker of endothelial differentiation, and increased levels of FGF-2 and FGFR-1 transcripts. LN-1 did not regulate signal transduction pathways known to operate down stream of FGF-2. Thus, phosphorylation of ERK was detected in FGF-2- but not in LN-1-treated cells. Taken together, this suggests that laminins may play a fundamental role in angiogenesis by directly affecting gene and protein expression profiles in endothelial cells.

Animals↗

PubMed2004

The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration.

Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor (VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGF-induced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner.

Actins↗

PubMed2004

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.

Animals↗

PubMed2004

A fragment of histidine-rich glycoprotein is a potent inhibitor of tumor vascularization.

In this study, we show that recombinant human histidine-rich glycoprotein (HRGP) has potent antiangiogenic properties as judged from effects on a syngeneic tumor model in C57/bl6 mice. Growth of fibrosarcoma, a very aggressive tumor, was reduced by >60% by HRGP treatment, and tumor angiogenesis was dramatically decreased. Treatment with HRGP led to increased apoptosis and reduced proliferation in the tumors. In contrast, HRGP did not affect apoptosis or DNA synthesis in endothelial cells or tumor cells in vitro. The mechanism of action of HRGP involves rearrangement of focal adhesions and decreased attachment of endothelial cells to vitronectin and, as a consequence, reduced endothelial cell migration. By using truncated versions of HRGP, we demonstrate that the isolated 150 amino acid-residue His/Pro-rich domain, which is also released by spontaneous proteolysis from purified HRGP, mediates the inhibitory effect on chemotaxis. Moreover, the His/Pro-rich domain must be released from HRGP to exert its effect. This study shows for the first time inhibitory effects of HRGP on tumor vascularization in vivo, thus providing proof of concept that HRGP is an angiogenesis inhibitor.

Angiogenesis Inhibitors↗

PubMed2004

Translocated in liposarcoma (TLS) is a substrate for fibroblast growth factor receptor-1.

Binding of fibroblast growth factor (FGF) to the high affinity receptor-1 (FGFR-1) leads to activation of its endogenous tyrosine kinase activity. A number of substrates for the FGFR-1 kinase have been identified. Among those, FGF receptor-substrate-2 (FRS-2) was identified by virtue of its interaction with p13suc, a yeast protein involved in cell cycle regulation. We have used immobilized p13suc to identify a new substrate for FGRF-1, which is identical to "translocated in liposarcoma" (TLS). TLS is a RNA/DNA-binding protein which occurs in fusion products with different transcription factors in a variety of solid tumours. We show that TLS is tyrosine phosphorylated in intact cells by a number of different growth factors, indicating a role in growth regulation.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Vascular endothelial growth factor receptor-3 in hypoxia-induced vascular development.

Reduced tissue oxygen tension (hypoxia) is appreciated as an efficient stimulus for neovascularization. The effect of hypoxia on the very first stages of vascular development is, however, less well characterized. Here we show that hypoxic conditions (1% O2) potently stimulated formation of an extensive vascular network during a discrete stage of mouse embryonal stem cell differentiation. The morphological changes correlated with an expanding pool of endothelial cells and with activation of the vascular endothelial growth factor-d (Vegf-d) and Vegf receptor-3 genes. VEGF receptor-3 expression was confined to vascular endothelial cells and analysis of the lymphatic marker Prox-1 revealed no expansion of lymphatic endothelial cells. Administration of neutralizing antibodies against either VEGF receptor-3 or VEGF receptor-2 impaired vascular network formation, whereas neutralizing antibodies against VEGF receptor-1 potentiated development of immature vascular structures. In addition, sequestering of VEGF receptor-3 ligands reduced vascularization in a manner similar to neutralization of VEGF receptor-3. We conclude that hypoxia-driven vascular development requires the activity of VEGF receptor-3.

Animals↗