Johan Kreuger

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.

Interactions between heparan sulfate and proteins : the concept of specificity

Proteoglycan (PG) coreceptors carry heparan sulfate (HS) chains that mediate interactions with growth factors, morphogens, and receptors. Thus, PGs modulate fundamental processes such as cell survival, division, adhesion, migration, and differentiation. This review summarizes recent biochemical and genetic information that sheds new light on the nature of HS–protein binding. Unexpectedly, many interactions appear to depend more on the overall organization of HS domains than on their fine structure.

Structural basis and potential role of heparin/ heparan sulfate binding to the angiogenesis inhibitor endostatin

Recombinant mouse endostatin produced by mammalian cells was shown to bind to heparin with a Kd of 0.3 μM, suggesting that this interaction may play a role in its anti‐angiogenic activity. Alanine mutagenesis demonstrated that a major site of four clustered arginines (positions 155, 158, 184 and 270) and a second site (R193,R194) are essential for binding. The same epitopes also participate in endostatin binding to heparan sulfate and sulfatides but not in its binding to the extracellular protein ligands fibulin‐1 and fibulin‐2. Analyses with various heparin fragments demonstrated a minimum size (12mer) for efficient binding to endostatin and a crucial role of 2‐O‐ and 6‐O‐sulfation. Furthermore, a substantial proportion (10–50%) of heparan sulfate chains obtained from various tissues showed a distinct binding to endostatin, indicating its potential to interact with extracellular and/or membrane‐bound proteoglycans. Angiogenesis induced by basic fibroblast growth factor‐2 (FGF‐2), but not by vascular endothelial growth factor (VEGF), in a chick chorioallantoic membrane assay could be inhibited by endostatin in a dose‐dependent manner. The mutational block of heparin binding decreased endostatin inhibition to low levels but elimination of zinc binding had no effect.

Inflammation-related genes up-regulated in schizophrenia brains

BackgroundMultiple studies have shown that brain gene expression is disturbed in subjects suffering from schizophrenia. However, disentangling disease effects from alterations caused by medication is a challenging task. The main goal of this study is to find transcriptional alterations in schizophrenia that are independent of neuroleptic treatment.MethodsWe compared the transcriptional profiles in brain autopsy samples from 55 control individuals with that from 55 schizophrenic subjects, subdivided according to the type of antipsychotic medication received.ResultsUsing global and high-resolution mRNA quantification techniques, we show that genes involved in immune response (GO:0006955) are up regulated in all groups of patients, including those not treated at the time of death. In particular, IFITM2, IFITM3, SERPINA3, and GBP1 showed increased mRNA levels in schizophrenia (p-values from qPCR ≤ 0.01). These four genes were co-expressed in both schizophrenic subjects and controls. In-vitro experiments suggest that these genes are expressed in both oligodendrocyte and endothelial cells, where transcription is inducible by the inflammatory cytokines TNF-α, IFN-α and IFN-γ.ConclusionAlthough the modified genes are not classical indicators of chronic or acute inflammation, our results indicate alterations of inflammation-related pathways in schizophrenia. In addition, the observation in oligodendrocyte cells suggests that alterations in inflammatory-related genes may have consequences for myelination. Our findings encourage future research to explore whether anti-inflammatory agents can be used in combination with traditional antipsychotics for a more efficient treatment of schizophrenia.

VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.

Endothelial Cell Migration in Stable Gradients of Vascular Endothelial Growth Factor A and Fibroblast Growth Factor 2 EFFECTS ON CHEMOTAXIS AND CHEMOKINESIS

Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 μm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.

Heparan sulfate biosynthesis: regulation and variability.

Nearly all vertebrate cells have been shown to express heparan sulfate proteoglycans (HSPGs) at the cell surface. The HSPGs bind to many secreted signaling proteins, including numerous growth factors, cytokines, and morphogens, to affect their tissue distribution and signaling. The heparan sulfate (HS) chains may have variable length and may differ with regard to both degree and pattern of sulfation. As the sulfation pattern of HS chains in most cases will determine if an interaction with a potential ligand will take place, as well as the affinity of the interaction, a key to understanding the function of HSPGs is to clarify how HS biosynthesis is regulated in different biological contexts. This review provides an introduction to the current understanding of HS biosynthesis and its regulation, and identifies research areas where more knowledge is needed to better understand how the HS biosynthetic machinery works.

Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function.

The anti‐angiogenic activity of endostatin (ES) depends on interactions with heparan sulfate (HS). In the present study, intact HS chains of ≥15 kDa bound quantitatively to ES whereas N‐sulfated HS decasaccharides, with affinity for several fibroblast growth factor (FGF) species, failed to bind. Instead, ES‐binding oligosaccharides composed of mixed N‐sulfated and N‐acetylated disaccharide units were isolated from pig intestinal HS. A 10/12mer ES‐binding epitope was identified, with two N‐sulfated regions separated by at least one N‐acetylated glucosamine unit (SAS‐domain). Cleavage at the N‐acetylation site disrupted ES binding. These findings point to interaction between discontinuous sulfated domains in HS and arginine clusters at the ES surface. The inhibitory effect of ES on vascular endothelial growth factor‐induced endothelial cell migration was blocked by the ES‐binding SAS‐domains and by heparin oligosaccharides (12mers) similar in length to the ES‐binding SAS‐domains, but not by 6mers capable of FGF binding. We propose that SAS‐domains modulate the biological activities of ES and other protein ligands with extended HS‐binding sites. The results provide a rational explanation for the preferential interaction of ES with certain HS proteoglycan species.

Fibroblast growth factors share binding sites in heparan sulphate

HS (heparan sulphate) proteoglycans bind secreted signalling proteins, including FGFs (fibroblast growth factors) through their HS side chains. Such chains contain a wealth of differentially sulphated saccharide epitopes. Whereas specific HS structures are commonly believed to modulate FGF-binding and activity, selective binding of defined HS epitopes to FGFs has generally not been demonstrated. In the present paper, we have identified a series of sulphated HS octasaccharide epitopes, derived from authentic HS or from biosynthetic libraries that bind with graded affinities to FGF4, FGF7 and FGF8b. These HS species, along with previously identified oligosaccharides that interact with FGF1 and FGF2, constitute the first comprehensive survey of FGF-binding HS epitopes based on carbohydrate sequence analysis. Unexpectedly, our results demonstrate that selective modulation of FGF activity cannot be explained in terms of binding of individual FGFs to specific HS target epitopes. Instead, different FGFs bind to identical HS epitopes with similar relative affinities and low selectivity, such that the strength of these interactions increases with increasing saccharide charge density. We conclude that FGFs show extensive sharing of binding sites in HS. This conclusion challenges the current notion of specificity in HS-FGF interactions, and instead suggests that a set of common HS motifs mediates cellular targeting of different FGFs.

Building blood vessels—stem cell models in vascular biology

Jakobsson et al. 2007. J. Cell Biol. doi:10.1083/jcb.200701146 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200701146%26rft_id%253Dinfo%253Apmid%252F17535968%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx