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1.
Microvasc Res ; 113: 65-70, 2017 09.
Article in English | MEDLINE | ID: mdl-28546076

ABSTRACT

ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA and the effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions. 15nM of ADAMTS-13 siRNA reduced HUVEC ADAMTS-13 protein levels by 90% after 24h incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. Furthermore, this transfection did not affect the HUVEC endogenous protein level of ADAMTS-1, a related family member of ADAMTS-13 indicating the specificity of the siRNA. Transfection of HUVEC with 15nM of ADAMTS-13 siRNA resulted in a 21% decrease in proliferation after 24h incubation. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24h after transfection with control siRNA, there was increased cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased protein levels of endogenous ADAMTS-13 also affected angiogenesis as measured by endothelial cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 40%. The protein level of vascular endothelial growth factor (VEGF), a well-known regulator of angiogenesis, was significantly decreased by 45% upon knockdown of ADAMTS-13. Moreover, activity of the AKT pathway, one of the VEGF angiogenesis downstream signaling pathways was down-regulated by ADAMTS-13 siRNA. These data indicate that in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through VEGF and AKT signaling pathway. Overall, our data suggest an additional model of endogenous ADAMTS-13 functionality, beyond that of cleaving von Willebrand factor.


Subject(s)
ADAMTS13 Protein/metabolism , Human Umbilical Vein Endothelial Cells/enzymology , Neovascularization, Physiologic , RNA Interference , RNA, Small Interfering/metabolism , ADAMTS13 Protein/genetics , Cell Movement , Cell Proliferation , Cells, Cultured , Down-Regulation , Humans , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , Signal Transduction , Time Factors , Transfection , Vascular Endothelial Growth Factor A/metabolism , von Willebrand Factor/metabolism
2.
Cell Mol Life Sci ; 72(2): 349-56, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24950743

ABSTRACT

Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. The goals of this study were to understand the angiogenic pathways ADAMTS13 activates and to identify the important structural components of ADAMTS13 that stimulate angiogenesis. Incubation of human umbilical vein endothelial cells (HUVEC) with 150 ng/mL (1 nM) of recombinant human ADAMTS13 induced VEGF expression by 53 % and increased VEGF mRNA by over sixfold, both within 10 min; the measured VEGF levels steadily decreased over 2 h, as shown by Western blot and ELISA. Phosphorylation of VEGFR2 was significantly enhanced in HUVEC after incubation with ADAMTS13 (1 nM). Structure-function analysis showed that an ADAMTS13 variant containing thrombospondin type 1 (TSP1) 2-8 repeats (TSP1 2-8), TSP1 2-8 plus CUB domains (TSP1 2-8 plus CUB), or TSP1 5-8 repeats plus CUB domains (TSP1 5-8 plus CUB) increased HUVEC proliferation by 41-54 % as compared to the EBM-2 controls. Chemotaxis assays further demonstrated that the TSP1 domains of ADAMTS13 increased HUVEC migration by 2.65-fold. Incubation of HUVEC with both ADAMTS13 variants containing TSP1 repeats and anti-VEGF IgG abrogated the enhanced effect of ADAMTS13 on proliferation, migration, and VEGFR2 phosphorylation. In conclusion, ADAMTS13-induced endothelial cell angiogenesis occurs via the upregulation of VEGF and phosphorylation of VEGFR2. This angiogenic activity depends on the C-terminal TSP1 repeats of ADAMTS13.


Subject(s)
ADAM Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Neovascularization, Physiologic/physiology , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , ADAMTS13 Protein , Blotting, Western , Cell Movement/physiology , Chemotaxis/physiology , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay , Human Umbilical Vein Endothelial Cells , Humans , Phosphorylation , Real-Time Polymerase Chain Reaction
3.
J Orthop Surg (Hong Kong) ; 21(1): 103-5, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23630000

ABSTRACT

Elderly people with osteoarthritis of the knee and postpartum women are at risk of insufficiency fractures of the proximal fibular or tibia. We report on an 82-year-old woman with valgus osteoarthritis of the knee who developed insufficiency fractures of the right proximal fibula and then the right proximal tibia. She underwent total knee arthroplasty using a long tibial stem and a tibial locking plate with the Less Invasive Stabilization System.


Subject(s)
Arthroplasty, Replacement, Knee , Fibula/injuries , Fractures, Stress/surgery , Osteoarthritis, Knee/surgery , Tibial Fractures/surgery , Aged, 80 and over , Female , Fractures, Stress/complications , Fractures, Stress/etiology , Humans , Osteoarthritis, Knee/complications , Tibial Fractures/complications , Tibial Fractures/etiology
4.
Microvasc Res ; 84(2): 109-15, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22626948

ABSTRACT

Plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. To assess the possible role of ADAMTS13 and its interactions with VEGF-mediated angiogenesis, the effects of ADAMTS13 on human umbilical vein endothelial cell (HUVEC) were studied in Matrigel tube formation, proliferation, cell migration, and scratch wound assays. Treatment of endothelial cells with exogenous recombinant full-length ADAMTS13 alone promoted angiogenesis in a dose-dependent manner. HUVEC incubated with 200 ng/mL ADAMTS13 (1.4 nM) resulted in a 65% increase in cell tube formation when compared to the EBM-2 control. HUVEC treated with 30 ng/mL ADAMTS13 (204.1 pM) resulted in an 83% increase in proliferation in a visual counting assay, whereas HUVEC treated with 10 ng/mL ADAMTS13 (68.0 pM) yielded a 295% increase in EC migration in a Boyden chamber assay. In contrast, ADAMTS13 inhibited VEGF-induced angiogenesis in a dose-dependent manner, with 200ng/mL inhibiting tube formation by 35%. HUVEC co-incubated with ADAMTS13 and an antibody to the ADAMTS13 thrombospondin domains 5-7 reversed the inhibition of tube formation. HUVEC treated with 30 ng/mL ADAMTS13 and 6.2 ng/mL (323.0 pM) VEGF proliferated 40% slower than the VEGF control after 24 h of incubation as measured by visual counting assay. Treatment of HUVEC with 6.2 ng/mL VEGF and 10 ng/mL ADAMTS13 inhibited cell migration by 48%, compared to the VEGF control. Substitution of ADAMTS13 with truncated ADAMTS13 (deletion of C-terminal TSP1 domain) did not significantly increase angiogenesis or suppress VEGF-induced angiogenesis, suggesting that the TSP1 domain is involved in ADAMTS13 angiogenic activities. Co-immunoprecipitation experiments provided further evidence that ADAMTS13 binds to VEGF via its TSP1 domain.


Subject(s)
ADAM Proteins/metabolism , Human Umbilical Vein Endothelial Cells/enzymology , Neovascularization, Physiologic , Vascular Endothelial Growth Factor A/metabolism , ADAM Proteins/antagonists & inhibitors , Antibodies/pharmacology , Binding Sites , Blotting, Western , Cell Movement , Cell Proliferation , Cells, Cultured , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Immunoprecipitation , Recombinant Proteins/metabolism , Time Factors
5.
Chem Commun (Camb) ; 47(37): 10455-7, 2011 Oct 07.
Article in English | MEDLINE | ID: mdl-21853179

ABSTRACT

A set of four phenylalanine analogues experiences diffusion retardation when transferred from phosphate-buffered saline into a peptide hydrogel of the same pH and ionic strength. The extent of retardation increases linearly with logP(oct), their lipophilicity.


Subject(s)
Hydrogels/chemistry , Oligopeptides/chemistry , Phenylalanine/chemistry , Amino Acid Sequence , Diffusion , Hydrophobic and Hydrophilic Interactions
6.
Org Lett ; 11(12): 2499-502, 2009 Jun 18.
Article in English | MEDLINE | ID: mdl-19441855

ABSTRACT

Cobalamin tethered to fluorescein or Rhodamine 6G has been synthesized and characterized. The fluorophore is conjugated to the ribose-5'-OH of cobalamin through a rigid linker to prevent the fluorophore from folding back through space and interacting with the corrin ring of cobalamin. This increases the fluorescence quantum yield. This new family of cobalamin analogues may be suitable for use as tumor markers to tag cancer cells for surgical resection.


Subject(s)
Biomarkers, Tumor/chemical synthesis , Cyclohexanes/chemistry , Fluorescent Dyes/chemical synthesis , Vitamin B 12/analogs & derivatives , Vitamin B 12/chemical synthesis , Biomarkers, Tumor/chemistry , Cell Line, Tumor/chemistry , Cell Line, Tumor/metabolism , DNA/chemistry , DNA/metabolism , Drug Design , Fluorescent Dyes/chemistry , Humans , Indicators and Reagents , Molecular Structure , Quantum Theory , Rhodamines/chemistry , Vitamin B 12/chemistry
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