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Primary lymphedema (PL), characterized by tissue swelling, fat accumulation, and fibrosis, results from defects in lymphatic vessels or valves caused by mutations in genes involved in development, maturation, and function of the lymphatic vascular system. Pathogenic variants in various genes have been identified in about 30% of PL cases. By screening of a cohort of 755 individuals with PL, we identified two TIE1 (tyrosine kinase with immunoglobulin- and epidermal growth factor-like domains 1) missense variants and one truncating variant, all predicted to be pathogenic by bioinformatic algorithms. The TIE1 receptor, in complex with TIE2, binds angiopoietins to regulate the formation and remodeling of blood and lymphatic vessels. The premature stop codon mutant encoded an inactive truncated extracellular TIE1 fragment with decreased mRNA stability, and the amino acid substitutions led to decreased TIE1 signaling activity. By reproducing the two missense variants in mouse Tie1 via CRISPR/Cas9, we showed that both cause edema and are lethal in homozygous mice. Thus, our results indicate that TIE1 loss-of-function variants can cause lymphatic dysfunction in patients. Together with our earlier demonstration that ANGPT2 loss-of-function mutations can also cause PL, our results emphasize the important role of the ANGPT2/TIE1 pathway in lymphatic function., Introduction The pathogenesis of primary lymphedema (PL) results from defective development and/or function of the lymphatic system (ref. 1; reviewed in refs. 2-5). PL is characterized by accumulation of interstitial [...]

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 p110a subunit or with smallmolecule 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., Introduction Research in recent years has greatly increased knowledge about lymphatic vessels and vessels with hybrid lymphatic and blood vascular markers, which are now considered to play active roles in [...]

Introduction Glaucoma is a group of heterogeneous diseases characterized by chronic, degenerative optic neuropathy with resultant loss of visual field (1). It is the second leading cause of blindness in [...], In glaucoma, aqueous outflow into the Schlemm's canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 2:2 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions. angiogenesis | lymphangiogenesis | vascular endothelial growth factor C | vascular endothelial growth factor receptor-2 www.pnas.org/cgi/doi/10.1073/pnas.0914318107

Author(s): Päivi Lindholm [1]; Merja H. Voutilainen [2]; Juha Laurén [1, 4]; Johan Peränen [1]; Veli-Matti Leppänen [1]; Jaan-Olle Andressoo [1]; Maria Lindahl [1]; Sanna Janhunen [2, 4]; Nisse Kalkkinen [...]

The zero-frequency vector control of an induction motor without a speed sensor has proven to be a very difficult task. Uncertainty and variation in the electrical parameters of the motor deteriorate the performance of fundamental-wave flux observers as the frequency approaches zero, and observers based on high-frequency signal injection rely on motor-specific phenomena. A new method has recently been proposed, where a low-frequency current signal is injected in the estimated rotor flux direction. If there is an error in the orientation, the signal gives rise to a torque oscillation and affects the back electromotive force (EMF) through the mechanical system. The phenomenon makes it possible to reach sensorless zero-frequency operation. This paper shows that the method is insensitive to the variation of the motor parameters. Experiments confirm the result. Index Terms--Induction motor drives, sensorless control.

In sensorless induction motor drives, flux estimators based only on the standard motor model work well at sufficiently high stator frequencies, but they fail at frequencies close to zero. To solve this problem, a new observer structure is proposed, combining a speed-adaptive full-order flux observer with a low-frequency signal-injection method. An error signal obtained from the signal-injection method is used as an additional feedback signal in the speed-adaptation law of the observer, resulting in a wide speed range, excellent dynamic properties, and zero-frequency operation capability. The enhanced observer is also robust against parameter errors. Experimental results are shown, including very slow speed reversals and long-term zero-frequency operation under rated load torque, as well as rated load torque steps and fast speed reversals under rated load torque. Index Terms--Flux estimation, induction motor drives, signal injection, speed sensorless.

A recently introduced speed-sensorless controller structure for tracking the rotor-flux orientation of an induction motor was implemented in a laboratory setup. The controller is based on superimposing a low-frequency ac test signal on the stator current of the motor. The response in the stator voltage to the signal depends on the orientation of the signal relative to that of the rotor flux. The dependency is due to the reaction of the mechanical system, and it is used to control the orientation error to zero. In the experiments, the controller operated successfully under nominal torque both at zero speed and at zero frequency. Slow and fast speed reversals under nominal torque were also successful. In addition, the system was capable of withstanding both motoring and generating load torque steps up to 50% of nominal torque. Index Terms--Induction motor drives, sensorless control.

An analysis of the decolorization of triphenyl methane, azo, polymeric and heterocyclic dyes by three purified lignin peroxidase isoenzymes from Phanerochaete chrysosporium and crude lignin peroxidase reveals that isoenzymes have distinct peculiarities toward dyes as substrates. Decolorezation is influenced greatly by lignin peroxidase, and manganese peroxidase is not needed to initiate the breaking down of the dyes. Three lignin peroxidase isoenzymes are LiP 4.15, LiP 4.65 and LiP 3.85.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor α1 (GFRa1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF2GFRα12 complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF2GFRα12 complex and the previously published artemin2GFRα32 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFRα interface between the two structures, in particular ones that buttress the key conserved ArgGFR&3X03B1;- Gluligand-ArgGFR&3X03B1; interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFRαs, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand2-co-receptor2-RET2 heterohexamer complexes. Consistent with this, we showed that GDNF2-GFRα12 and artemin2-GFRα32 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFRα1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg190-Lys202 region in GFR&3X03B1;1 domain 2. This may explain how GDNFGFRα1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET. [ABSTRACT FROM AUTHOR]

The structure of inactive pyruvate formate-lyase in complex with a natural substrate, pyruvate, was solved at 2.7 Å resolution. Both active sites of the homodimeric enzyme are occupied by pyruvate; additional binding sites were not found. Pyruvate was found in a cleft close to the active-site cysteines 418 and 419, with the carboxyl group in contact with arginines 176 and 435 and the methyl group within van der Waals distance of Phe327. It is believed that the binding site of pyruvate is not the position of pyruvate as the reaction initiates, as conformational changes occur during activation of the enzyme. [ABSTRACT FROM AUTHOR]

Vascular bypass procedures in the central nervous system (CNS) remain technically challenging, hindered by complications and often failing to prevent adverse outcome such as stroke. Thus, there is an unmet clinical need for a safe and effective CNS revascularization. Vascular endothelial growth factors (VEGFs) are promising candidates for revascularization; however, their effects appear to be tissuespecific and their potential in the CNS has not been fully explored. To test growth factors for angiogenesis in the CNS, we characterized the effects of endothelium-specific growth factors on the brain vasculature and parenchyma. Recombinant adeno-associated virus (AAV) vectors encoding the growth factors were injected transcranial to the frontoparietal cerebrum of mice. Angiogenesis, mural cell investment, leukocyte recruitment, vascular permeability, reactive gliosis and neuronal patterning were evaluated by 3-dimensional immunofluorescence, electron microscopy, optical projection tomography, and magnetic resonance imaging. Placenta growth factor (PIGF) stimulated robust angiogenesis and arteriogenesis without significant side effects, whereas VEGF and VEGF-C incited growth of aberrant vessels, severe edema, and inflammation. VEGF-B, angiopoietin-1, angiopoietin-2, and a VEGF/ angiopoietin-1 chimera had minimal effects on the brain vessels or parenchyma. Of the growth factors tested, PIGF emerged as the most efficient and safe angiogenic factor, hence making it a candidate for therapeutic CNS revascularization [ABSTRACT FROM AUTHOR]

VEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 lg-homology domains; domains 2 and 3 (D23) represent the ligandbinding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation. In the present study, isothermal titration calorimetry showed that the Gibbs free energy of VEGF-A, VEGF-C, or VEGF-E binding to 023 or the full-length ECD of VEGFR-2 is dominated by favorable entropic contribution with enthalpic penalty. The free energy of VEGF binding to the ECD is 1.0-1.7 kcallmol less favorable than for binding to D23. A model of the VEGF-EI VEGFR-2 ECD complex derived from smallangle scattering data provided evidence for homotypic interactions in D4-7. We also solved the crystal structures of complexes between VEGF-A or VEGF-E with 023, which revealed comparable binding surfaces and similar interactions between the ligands and the receptor, but showed variation in 023 twist angles. The energetically unfavorable homotypic interactions in D4-7 may be required for re-orientation of receptor monomers, and this mechanism might prevent ligand-independent activation of VEGFR-2 to evade the deleterious consequences for blood and lymph vessel homeostasis arising from inappropriate receptor activation. [ABSTRACT FROM AUTHOR]