Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation; however, little is known about the structure-function relationships of Ang1 with its receptor, Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2). In this study, we generated several Ang1 and angiopoietin-2 (Ang2) variants to define the role of the superclustering and oligomerization domains of the Ang1 protein. Then we analyzed the molecular structure of the variants with SDS-PAGE and rotary metal-shadowing transmission electron microscopy (RMSTEM) and determined the effects of these variants on the binding and activation of Tie2. Ang1 exists as heterogeneous multimers with basic trimeric, tetrameric, and pentameric oligomers, whereas Ang2 exists as trimeric, tetrameric, and pentameric oligomers. The variant Ang1C265S, consisting of trimers, tetramers, and pentamers without multimeric forms of Ang1, yielded less Tie2 activation than did Ang1, whereas monomeric Ang1 (Ang1/FD), dimeric Ang1 variants (Ang1D2, and Ang1D3), and dimeric and trimeric Ang1 variant (Ang1D1) dramatically lost their ability to bind and activate Tie2. An Ang1 protein in which two cysteines (amino acids 41 and 54) were replaced with serines (Ang1C41S/C54S) formed mostly dimers and trimers that were not able to bind and activate Tie2. In addition, improper creation of a new cysteine in Ang2 (Ang2S263C) dramatically induced Ang2 aggregation without activating Tie2. In conclusion, proper oligomerization of Ang1 having at least four subunits by the intermolecular disulfide linkage involving cysteines 41 and 54 is critical for Tie2 binding and activation. Thus, our data shed a light on the structurefunction relationships of Ang1 with Tie2.Angiopoietin-1 (Ang1) 1 was discovered as a secreted protein ligand of tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2Ј (Tie2) (1). Tie2 is a member of the receptor tyrosine kinase family and is expressed predominantly on vascular endothelial cells and early hematopoietic cells (2-4). Ang1-and Tie2-deficient mice have similar phenotypes characterized by embryonic lethality with severe defects in vascular remodeling, insufficient vessel stabilization, and perturbed vascular maturation, indicating that Ang1 and Tie2 play critical roles in vascular development (5, 6). Accordingly, transgenic overexpression or gene transfer of Ang1 not only enhances vessel formation, but also protects the adult vasculature against vascular leakage (7-10). In addition, Ang1 can counteract vascular endothelial growth factor-induced side effects (9, 11) while having an additive effect on vessel formation (8, 10). Thus, Ang1 is a very promising growth factor for therapeutic angiogenesis (12, 13). Moreover, in a series of experiments, we found that the Ang1/Tie2 system in normal adult blood vessels was important in maintaining the integrity of nonproliferating endothelial cells by strongly inducing endothelial cell survival against insult-mediated damage (14 -16). Furthermore, recent series o...

Interaction between ephrinB2 and EphB4 in endothelial cells at the arterial-venous capillary interface is critical for proper embryonic capillary morphogenesis. However, the intracellular downstream signaling of ephrinB2-EphB in vascular endothelial cells is unknown. This study examined the effect of ephrinB2-induced activation of EphB kinases on vascular endothelial growth factor (VEGF)- and angiopoietin-1 (Ang1)-induced Ras/mitogen-activated protein kinase (MAPK) signaling cascades in human umbilical vein endothelial cells (HUVECs). Reverse transcriptase-polymer chain reaction results showed that HUVECs expressed three kinds of EphB kinases known to bind to ephrinB2: EphB2, EphB3, and EphB4. EphrinB2 not only increased the phosphorylation of EphB2 and EphB4 in a time-dependent manner but also increased recruitment of p120-Ras-GTPase-activating protein (p120-RasGAP) to EphB2 and EphB4. Accordingly, ephrinB2 inhibited VEGF- and Ang1-induced Ras-MAPK activities, whereas ephrinB2 did not alter VEGF-induced Flk phosphorylation or Ang1-induced Tie2 phosphorylation. Furthermore, ephrinB2 suppressed VEGF- and Ang1-induced proliferation and/or migration, which are mediated mainly through Ras/MAPK signaling cascades. From these results, we propose that ephrinB2-EphB, signaling through Ras/MAPK cascade, may be critical for proper morphogenesis of capillary endothelium through the arrest of endothelial cell proliferation and migration at the arterial-venous interface.

The angiopoietin (Ang) family of growth factors includes Ang1, Ang2, Ang3, and Ang4, all of which bind to the endothelial receptor tyrosine kinase Tie2. Ang3 (mouse) and Ang4 (human) are interspecies orthologs. In experiments with human endothelial cell lines, Ang3 was identified as an antagonist of Tie2 and Ang4 was identified as an agonist of Tie2. However, the biological roles of Ang3 and Ang4 are unknown. We examined the biological effect of recombinant Ang3 and Ang4 proteins in primary cultured endothelial cells and in vivo in mice. Recombinant Ang3 and Ang4 formed disulfide-linked dimers. Ang4 (400 ng/mL) markedly increased Tie2 and Akt phosphorylation in primary cultured HUVECs whereas Ang3 (400 ng/mL) did not produce significant changes. Accordingly, Ang4, but not Ang3, induced survival and migration in primary cultured HUVECs. Unexpectedly, intravenously administered Ang3 (30 microg) was more potent than Ang4 (30 microg) in phosphorylating the Tie2 receptor in lung tissue from mice in vivo. Accordingly, Ang3 was more potent than Ang4 in phosphorylating Akt in primary cultured mouse lung microvascular endothelial cells. Ang3 and Ang4 both produced potent corneal angiogenesis extending from the limbus across the mouse cornea in vivo. Thus, Ang3 and Ang4 are agonists of Tie2, but mouse Ang3 has strong activity only on endothelial cells of its own species.

Fractalkine is an unusual tumor necrosis factor (TNF)-alpha-induced chemokine. The molecule is tethered to cells that express it and produces strong and direct adhesion to leukocytes expressing fractalkine receptor. However, the potential mechanism and significance of TNF-alpha-induced fractalkine expression in vascular endothelial cells are poorly understood. Here we show that in primary cultured endothelial cells TNF-alpha-induced fractalkine mRNA expression is mediated mainly through phosphatidylinositol 3'-kinase activation and nuclear factor (NF)-kappaB mediated transcriptional activation, along with GC-rich DNA-binding protein-mediated transcription. Interestingly, GC-rich DNA-binding protein inhibitors, mithramycin A and chromomycin A3, strongly suppressed TNF-alpha-induced fractalkine mRNA expression, possibly through inhibition of transcriptional activities by NF-kappaB and Sp1. In fact, direct inhibition of NF-kappaB and Sp1 bindings by decoy oligonucleotides suppressed TNF-alpha-induced fractalkine expression. Histologically, TNF-alpha-induced fractalkine expression was observed markedly in arterial and capillary endothelial cells, endocardium, and endothelium of intestinal villi, and slightly in venous endothelial cells, but not at all in lymphatic endothelial cells of intestine. Mithramycin A markedly suppressed TNF-alpha-induced fractalkine expression in vivo. These results indicate that TNF-alpha-stimulated fractalkine expression could act as part of arterial endothelial adhesion to leukocytes and monocytes during inflammation and atherosclerosis. NF-kappaB and Sp1 inhibitors such as mithramycin A may provide a pharmacological approach to suppressing these processes.

Abstract-Fractalkine is a unique chemokine that functions as a chemoattractant as well as an adhesion molecule on endothelial cells activated by proinflammatory cytokines. Alpha-lipoic acid (LA), a naturally occurring dithiol compound, is an essential cofactor for mitochondrial bioenergetic enzymes. LA improves glycemic control, reduces diabetic polyneuropathies, and mitigates toxicity associated with heavy metal poisoning. The effects of LA on processes associated with sepsis, however, are unknown. We evaluated the antiinflammatory effect of LA on fractalkine expression in a lipopolysaccharide-induced endotoxemia model. Tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ (IL-1␤) significantly induced fractalkine mRNA and protein expression in endothelial cells. LA strongly suppressed TNF-␣-or IL-1␤-induced fractalkine expression in endothelial cells by suppressing the activities of nuclear factor-B and specificity protein-1. LA also decreased TNF-␣-or IL-1␤-stimulated monocyte adhesion to human umbilical vein endothelial cells. As shown by immunohistochemistry, fractalkine protein expression was markedly increased by treatment with lipopolysaccharide in arterial endothelial cells, endocardium, and endothelium of intestinal villi. LA suppressed lipopolysaccharide-induced fractalkine protein expression and infiltration of endothelin 1-positive cells into the heart and intestine in vivo. LA protected against lipopolysaccharide-induced myocardial dysfunction and improved survival in lipopolysaccharide-induced endotoxemia. These results suggest that LA could be an effective agent to reduce fractalkine-mediated inflammatory processes in endotoxemia. Key Words: ␣-lipoic acid Ⅲ fractalkine Ⅲ endothelial cells Ⅲ inflammation S epsis is a clinical syndrome that represents the systemic response to an infection and is characterized by systemic inflammation and widespread tissue injury. At the site of injury, the endothelium expresses various adhesion molecules that attract leukocytes. 1 At the same time, inflammatory cells are activated and express a variety of adhesion molecules that cause the aggregation and margination of these cells to the vascular endothelium. 2 When the inflammatory response is initiated, a wide variety of chemical mediators are released into circulation. These chemical mediators, including tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ (IL-1␤), are associated with the continuation of the inflammatory response. 3 Sepsis is caused mainly by an exaggerated systemic response to endotoxemia induced by gram-negative bacteria and their characteristic cell wall component, lipopolysaccharide (LPS). 4 In mice, challenge with high doses of LPS results in a syndrome resembling septic shock in humans. 5 Fractalkine (CX3CL1) is a structurally novel protein in which a soluble chemokine-like domain is fused to a mucin stalk that extends into the cytoplasm across the cell membrane. 6 Fractalkine is expressed in activated endothelial cells, and its expression is upregulated by TNF-␣, IL-1␤, and LPS. 7,8 As a full-...

ObjectiveTo identify the effects of a custom-made rigid foot orthosis (RFO) in children over six years old with pes planus.MethodsThe medical records of 39 children (mean age, 10.3±4.09 years) diagnosed with pes planus, fitted with RFOs, and had who more than two consecutive radiological studies were reviewed. The resting calcaneal stance position (RCSP), anteroposterior talocalcaneal angle (APTCA), lateral talocalcaneal angle (LTTCA), the lateral talometatarsal angle (LTTMA), and calcaneal pitch (CP) of both feet were measured to evaluate foot alignment. After diagnosis, children were fitted with a pair of RFOs and recommended to walk with heel strike and reciprocal arm swing to normalize the gait pattern. A follow-up clinical evaluation with radiological measurements was performed after 12-18 months and after 24 months of RFO application. Post-hoc analysis was used to test for significant differences between the radiological indicators and RCSP.ResultsWith RFOs, all radiological indicators changed in the corrective direction except LTTCA. RCSP and CP in the third measurement showed significant improvement in comparison with the second and baseline measurements. Additionally, APTCA and LTTMA revealed improvements at the third measurement versus the baseline measurements.ConclusionThis study revealed that radiological indicators improved significantly after 24 months of RFO application. A prospective long-term controlled study with radiographical evaluation is necessary to confirm the therapeutic effects of RFOs and to determine the optimal duration of wear in children with pes planus.

Although orthotic modification using the inverted technique is available for the treatment of flatfoot, empirical evidence for the biomechanical effects of inverted-angle foot orthoses (FOs) is lacking. The aim of this study was to evaluate the effects of different FO inversion angles on plantar pressure during gait in children with flatfoot. Twenty-one children with flexible flatfeet (mean age 9.9 years) were enrolled in this study. The plantar pressures were measured for the rearfoot; medial and lateral midfoot; and medial, central, and lateral forefoot as participants walked on a treadmill while wearing shoes only and shoes with the following 3 orthotic conditions: (i) orthosis with no inverted angle, (ii) orthosis with a 15° inverted angle, and (iii) orthosis with a 30° inverted angle. A one-way repeated measures analysis of variance (ANOVA) with the Bonferroni-adjusted post-hoc test was used to compare the mean values of each orthotic condition. Compared with the shoe only condition, the peak pressure decreased significantly under the medial forefoot and rearfoot with all FOs (p <0.05). However, no significant differences in the peak pressure under the medial forefoot and rearfoot were observed between the FOs. The peak pressure under the medial midfoot increased significantly with all FOs, and a maximal increase in the peak pressure was obtained with a 30° inverted angle orthosis. Furthermore, the contact area under the medial midfoot and rearfoot increased significantly with all FOs, compared with the shoe only condition (p <0.05). Again, no significant differences were observed between the FOs. For plantar pressure redistribution, a FO with a low inverted angle could be effective, accommodative, and convenient for children with flatfoot.

The loss of an upper limb significantly limits the functional activities of daily living. A huge emphasis is placed on the manipulation, shape, weight, and comfort of a prosthesis, to enable its use as an inherent body part. Even with technological advances, customized upper-extremity myoelectric prosthesis remain heavy and expensive. The high cost of upper-extremity prosthesis is an especially steep economic barrier for patients. Three-dimensional (3D) printing is a promising avenue for reducing the cost of prosthesis. We applied 3D-printed pressure-sensored prosthetics to a traumatic transradial amputee, and compared the hand functions with a customized myoelectric prosthesis. The 3D-printed pressure-sensored prosthetics showed low grip strength and decreased dexterity compared to the conventional myoelectric prosthesis. Although there were a few limitations, the fabrication of prosthesis with 3D printing technology can overcome previous problems such as high production cost, long fabrication period and heavy weight.