Peroxidasin is essential for endothelial cell survival and growth signaling by sulfilimine crosslink-dependent matrix assembly.

Peroxidasin (PXDN) has been reported to crosslink the C-terminal non-collagenous domains of collagen IV (Col IV) by forming covalent sulfilimine bond. Here, we explored the physiological role of PXDN and its mechanism of action in endothelial cell survival and growth. Silencing of PXDN using siRNAs decreased cell proliferation without increase of the number of detached cells and decreased cell viability under serum-starved condition with increased fragmented nuclei and caspase 3/7 activity. Conditioned medium (CM) containing wild-type PXDN restored the proliferation of PXDN-depleted cells, but CM containing mutant PXDN with deletion of either N-terminal extracellular matrix (ECM) motifs or peroxidase domain failed to restore PXDN function. Accordingly, anti-PXDN antibody [raised against IgC2 (3-4) subdomain within ECM motifs] and peroxidase inhibitor phloroglucinol prevented the rescue of the PXDN-depleted cells by PXDN-containing CM. PXDN depletion resulted in loss of sulfilimine crosslinks, and decreased dense fibrillar network assembly of not only Col IV, but also fibronectin and laminin like in Col IV knockdown. Exogenous PXDN-containing CM restored ECM assembly as well as proliferation of PXDN-depleted cells. Accordingly, purified recombinant PXDN protein restored the proliferation and ECM assembly, and prevented cell death of the PXDN-depleted cells. PXDN depletion also showed reduced growth factors-induced phosphorylation of FAK and ERK1/2. In addition, siPXDN-transfected cell-derived matrix failed to provide full ECM-mediated activation of FAK and ERK1/2. These results indicate that both the ECM motifs and peroxidase activity are essential for the cellular function of PXDN and that PXDN is crucial for ECM assembly for survival and growth signaling.

A Subset of Paracrine Factors as Efficient Biomarkers for Predicting Vascular Regenerative Efficacy of Mesenchymal Stromal/Stem Cells.

Mesenchymal stromal/stem cells (MSCs) have been developed as a promising source for cell-based therapies of ischemic disease. However, there are some hurdles in their clinical application such as poor cell engraftment and inconsistent stem cell potency. In this study, we sought to find biomarkers for predicting potency of MSCs for proangiogenic therapy to improve their beneficial effects. Large variations were observed in proangiogenic factor secretion profiles of conditioned media derived from nine different donor-derived Wharton's jelly (WJ)-derived MSCs and 8 factors among 55 angiogenesis-related factors were secreted at considerable levels. Two distinct WJ-MSCs that had the lowest or the highest secretion of these eight factors showed corresponding proangiogenic activities in in vitro angiogenesis assays. When four additional different donor-derived WJ-MSCs were further examined, proangiogenic activities in migration and tube formation of endothelial cells and in in vivo Matrigel plug assay were highly consistent with secretion levels of four major factors (angiogenin, interleukin-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor). Such correlation was also observed in vascular regenerative effect in a mouse hind limb ischemia model. Blocking of these four factors by neutralizing antibodies or knockdown of them by siRNA treatment resulted in significant inhibition of proangiogenic activities of not only WJ-MSCs, but also bone marrow-derived MSCs. These results suggest that these four factors may represent efficient biomarkers for predicting vascular regenerative efficacy of MSCs. Stem Cells 2019;37:77-88.

Biallelic Deletion of Pxdn in Mice Leads to Anophthalmia and Severe Eye Malformation.

Peroxidasin (PXDN) is a unique peroxidase containing extracellular matrix motifs and stabilizes collagen IV networks by forming sulfilimine crosslinks. PXDN gene knockout in Caenorhabditis elegans (C. elegans) and Drosophila results in the demise at the embryonic and larval stages. PXDN mutations lead to severe eye disorders, including microphthalmia, cataract, glaucoma, and anterior segment dysgenesis in humans and mice. To investigate how PXDN loss of function affects organ development, we generated Pxdn knockout mice by deletion of exon 1 and its 5' upstream sequences of the Pxdn gene using the CRISPR/Cas9 system. Loss of both PXDN expression and collagen IV sulfilimine cross-links was detected only in the homozygous mice, which showed completely or almost closed eyelids with small eyes, having no apparent external morphological defects in other organs. In histological analysis of eye tissues, the homozygous mice had extreme defects in eye development, including no eyeballs or drastically disorganized eye structures, whereas the heterozygous mice showed normal eye structure. Visual function tests also revealed no obvious functional abnormalities in the eyes between heterozygous mice and wild-type mice. Thus, these results suggest that PXDN activity is essential in eye development, and also indicate that a single allele of Pxdn gene is sufficient for eye-structure formation and normal visual function.

Autophagy mediates enhancement of proangiogenic activity by hypoxia in mesenchymal stromal/stem cells.

Mesenchymal stromal/stem cells (MSCs) have been promising source for regenerative cell therapy in ischemic diseases. To improve efficacy of MSC therapy, various priming methods have been developed, and hypoxic priming has been reported to enhance therapeutic efficacy of MSCs by increasing secretion level of growth factors and cytokines. Recently, it has been reported that bone marrow MSCs primed with hypoxic condition show an increase of autophagy. Here, we addressed whether proangiogenic activity increased by hypoxic condition is associated with autophagy. Wharton's jelly derived MSCs primed with hypoxia showed increase of autophagy with increased hypoxia inducible factor-1α level, and conditioned medium (CM) derived from these cells showed increased levels of migration and tube formation of human umbilical vein endothelial cells (HUVECs) compared to non-primed MSCs-derived CM. Pretreatment with autophagy inhibitor 3-methyladenine or chloroquine prior to exposure of hypoxia resulted in reduction of migration and tube formation of HUVECs. CM obtained under hypoxic condition from MSCs in which autophagy activity was inhibited by ATG5 and ATG7 siRNA treatment also showed decrease of migration and tube formation of HUVECs. Accordingly, secretion levels of angiogenin and VEGF that were markedly increased upon hypoxia exposure was decreased by ATG5/7 knockdown. Therefore, it may be suggested that autophagy plays an important role in hypoxia-driven enhancement of paracrine effect of MSCs.

A Novel Peptide Derived From Tissue-Type Plasminogen Activator Potently Inhibits Angiogenesis and Corneal Neovascularization.

The recombinant protein TK1-2, which consists of two kringle domains of tissue-type plasminogen activator (t-PA), inhibits angiogenesis and tumor growth. ɪn this study, we examined the anti-angiogenic activities of peptides derived from kringle 2 domain of t-PA to identify the functional core sequence. Seven peptides were constructed from the kringle 2 sequence, based on the structure and characteristics of amino acid residues, and were analyzed for their inhibitory effects on endothelial cells (ECs). Among them, TP-7 (derived from a ß-sheet motif) potently inhibited proliferation, tube formation, and migration of ECs in a dose-dependent manner, whereas truncation of 3-9 amino acid residues from either N or C terminus of TP-7 abrogated its inhibitory effects on ECs. TP-7 also potently inhibited angiogenesis in a Matrigel plug assay in vivo. Moreover, TP-7 dose-dependently suppressed corneal neovascularization induced by an acute chemical burn in a rat model. At the molecular level, TP-7 inhibited VEGF- or bFGF-induced phosphorylation of FAK and ERK1/2 and drastically disrupted VEGF- or bFGF-induced formation of stress fibers and focal adhesion complexes. In addition, TP-7 markedly suppressed attachment and spreading of ECs on a collagen type I or fibronectin matrix. Adhesion of ECs to immobilized TP-7 increased dose-dependently, which was disrupted strongly by pretreatment with soluble TP-7 and slightly by an integrin α2ß1-blocking antibody. These results suggest that TP-7 is a potent anti-angiogenic peptide in part affecting the integrin α2ß1-dependent pathway and that it can be used for treatment of corneal neovascularization by targeting VEGF and non-VEGF pathways. J. Cell. Biochem. 118: 1132-1143, 2017. © 2016 Wiley Periodicals, Inc.

Priming Wharton's jelly-derived mesenchymal stromal/stem cells with ROCK inhibitor improves recovery in an intracerebral hemorrhage model.

Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron-like cells under specific conditions and to secrete paracrine factors for neuroprotection and regeneration. Previously, Rho-kinase inhibitors have been reported to potentiate differentiation of rodent bone marrow MSCs into neuron-like cells induced by CoCl2 (HIF-1α activation-mimicking agent). Here, a strategy of priming MSCs with fasudil, a Rho-kinase inhibitor, was investigated using Wharton's jelly-derived MSCs (WJ-MSCs) to improve recovery in a rat model of intracranial hemorrhage (ICH). In vitro culture of WJ-MSCs by co-treatment with fasudil (30 µM) and CoCl2 provoked morphological changes of WJ-MSCs into neuron-like cells and increased the expression of neuronal markers. Assessment of the secretion profiles showed that fasudil (30 µM) specifically increased glial cell line-derived neurotrophic factor (GDNF) among the secreted proteins at the transcription and secretion levels. For in vivo experiments, WJ-MSCs primed with fasudil (10 µM, exposure for 6 h) were transplanted into ICH rats with HIF-1α upregulation 1 week after injury, and neurological function was assessed via rotarod and limb placement tests for 7 weeks after transplantation. The group with WJ-MSCs primed with fasudil showed improved functional performance compared with the non-primed group. Accordingly, the primed group showed stronger expression of GDNF and higher levels of microtubule-associated protein 2 and neurofilament-H positive-grafted cells in the ICH lesion 3 weeks after transplantation compared with the non-primed group. Therefore, this work suggests that priming WJ-MSCs with fasudil is a possible application for enhanced cell therapy in stroke, with additional beneficial effect of up-regulation of GDNF.

Enhancing Anti-Cancer Therapy with Selective Autophagy Inhibitors by Targeting Protective Autophagy.

Autophagy is a process of eliminating damaged or unnecessary proteins and organelles, thereby maintaining intracellular homeostasis. Deregulation of autophagy is associated with several diseases including cancer. Contradictory dual roles of autophagy have been well established in cancer. Cytoprotective mechanism of autophagy has been extensively investigated for overcoming resistance to cancer therapies including radiotherapy, targeted therapy, immunotherapy, and chemotherapy. Selective autophagy inhibitors that directly target autophagic process have been developed for cancer treatment. Efficacies of autophagy inhibitors have been tested in various pre-clinical cancer animal models. Combination therapies of autophagy inhibitors with chemotherapeutics are being evaluated in clinal trials. In this review, we will focus on genetical and pharmacological perturbations of autophagy-related proteins in different steps of autophagic process and their therapeutic benefits. We will also summarize combination therapies of autophagy inhibitors with chemotherapies and their outcomes in pre-clinical and clinical studies. Understanding of current knowledge of development, progress, and application of cytoprotective autophagy inhibitors in combination therapies will open new possibilities for overcoming drug resistance and improving clinical outcomes.

The recombinant kringle domain of urokinase plasminogen activator inhibits VEGF165-induced angiogenesis of HUVECs by suppressing VEGFR2 dimerization and subsequent signal transduction.

The recombinant kringle domain (UK1) of urokinase plasminogen activator was previously reported to exert antiangiogenic activity against Vascular Endothelial Growth Factor (VEGF)-induced angiogenesis in both in vitro and in vivo models. In this study, we explored the molecular signaling mechanisms involved in the antiangiogenic activity of UK1 by examining VEGF signaling proteins. VEGF165 stimulates the phosphorylation of VEGF signaling molecules, and pretreatment with UK1 blocked VEGF-induced signal transduction associated with proliferation, survival, and migration. UK1 also suppressed VEGF165-induced activation of MMP-2. Moreover, UK1 suppressed the phosphorylation and activation of VEGFR2 in VEGF-stimulated human umbilical cord vein endothelial cells (HUVECs) by blocking the dimerization of VEGFR2. Overall, our findings suggest that UK1 inhibits VEGF-induced proliferation, migration, and matrix metalloproteinase activity of HUVECs by suppressing VEGFR2 dimerization and subsequent angiogenic signals.

Efficient nonadhesive ex vivo expansion of early endothelial progenitor cells derived from CD34+ human cord blood fraction for effective therapeutic vascularization.

Endothelial progenitor cells (EPCs) have been shown to have therapeutic potential in ischemic disease. However, the number of EPCs for cell therapy is limited. In this study, instead of the typical adherent culture method, we investigated a more efficient, clinically applicable nonadhesive expansion method for early EPCs using cord blood-derived cells to overcome rapid cellular senescence. After a suspension culture of isolated CD34(+) cells in serum-free medium containing each cytokine combination was maintained for 9 d, the number of expanded functional EPCs was assessed by an adherent culture assay. Compared to mononuclear cells, the CD34(+) fraction was superior in its expansion of functional EPCs that could differentiate into acLDL/UEA-1(+) cells without significant cellular senescence, whereas the CD34(-) fraction showed no EPC expansion. Among the cytokine combinations tested for the CD34(+) fraction, a combination (SFIb) consisting of stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand, interleukin-3, and basic fibroblast growth factor resulted in a reproducible 64- to 1468-fold EPC expansion from various cord blood origins. Interestingly, the SFIb combination displayed markedly increased EPC expansion (2.43-fold), with a higher percentage of CD34(+) cells (2.17-fold), undifferentiated blasts (2.38-fold) and CXCR4(+) cells (1.68-fold) compared to another cytokine combination (SCF, thrombopoietin, and granulocyte colony-stimulating factor), although the two cytokine combinations had a similar level of total mononucleated cell expansion (∼ 10% difference). Accordingly, the cells expanded in the SFIb combination were more effective in recovery of blood flow and neovascularization in hind-limb ischemia in vivo. Taken together, these results suggest that the nonadhesive serum-free culture conditions of the CD34(+) fraction provide an effective EPC expansion method for cell therapy, and an expansion condition leading to high percentages of CD34(+) cells and blasts is likely important in EPC expansion.

Enhancement of endothelial progenitor cell numbers and migration by H1152, a Rho kinase specific inhibitor.

Endothelial progenitor cells (EPCs) are applied in the treatment of ischemic diseases. In ex vivo culture of human cord-blood derived EPCs, H1152, (S)-(+)-2-methyl-1-[(4-methyl-5-iso-quinolinyl) sulfonyl]-homopiperazine, markedly increased the number of EPCs. It also induced EPC migration, stimulated the phosphorylation of AKT, and reduced the expression of p27 in the EPCs. Thus H1152 can be used effectively in ex vivo expansion of EPCs.

Modulation of Autophagy is a Potential Strategy for Enhancing the Anti-Tumor Effect of Mebendazole in Glioblastoma Cells.

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has been suggested as a repositioning candidate for the treatment of brain tumors. However, the efficacy of MBZ needs further study to improve the beneficial effect on the survival of those patients. In this study, we explored a novel strategy to improve MBZ efficacy using a drug combination. When glioblastoma cells were treated with MBZ, cell proliferation was dose-dependently inhibited with an IC50 of less than 1 µM. MBZ treatment also inhibited glioblastoma cell migration with an IC50 of less than 3 µM in the Boyden chamber migration assay. MBZ induced G2-M cell cycle arrest in U87 and U373 cells within 24 h. Then, at 72 h of treatment, it mainly caused cell death in U87 cells with an increased sub-G1 fraction, whereas polyploidy was seen in U373 cells. However, MBZ treatment did not affect ERK1/2 activation stimulated by growth factors. The marked induction of autophagy by MBZ was observed, without any increased expression of autophagy-related genes ATG5/7 and Beclin 1. Co-treatment with MBZ and the autophagy inhibitor chloroquine (CQ) markedly enhanced the anti-proliferative effects of MBZ in the cells. Triple combination treatment with temozolomide (TMZ) (another autophagy inducer) further enhanced the anti-proliferative effect of MBZ and CQ. The combination of MBZ and CQ also showed an enhanced effect in TMZ-resistant glioblastoma cells. Therefore, we suggest that the modulation of protective autophagy could be an efficient strategy for enhancing the anti-tumor efficacy of MBZ in glioblastoma cells.

N,N-Dimethyl phytosphingosine sensitizes HL-60/MX2, a multidrug-resistant variant of HL-60 cells, to doxorubicin-induced cytotoxicity through ROS-mediated release of cytochrome c and AIF.

Doxorubicin (Dox) is widely used to treat a variety of tumors. However, resistance to this drug is common, making successful treatment more difficult. Previously, we introduced a novel phytosphingosine derivative, N,N-dimethyl phytosphingosine (DMPS), as a potent anticancer therapeutic agent in human leukemia cells. This study was performed to investigate whether DMPS can sensitize HL-60/MX2, a multidrug-resistant variant of HL-60, to Dox-induced apoptosis. Low concentrations of DMPS sensitized HL-60/MX2 cells to Dox-induced apoptosis. Combined Dox + DMPS treatment-induced apoptosis was accompanied by the activation of caspase-8 and caspase-3 as well as PARP cleavage. Cytochrome c and AIF release were also observed in Dox + DMPS-treated HL60/MX2 cells. Pretreatment with z-VAD-fmk markedly prevented caspase-3 activation and moderately suppressed apoptosis, suggesting that Dox + DMPS-induced apoptosis is somewhat (not completely) dependent on caspase. Cytochrome c and AIF release were not affected by pretreatment with z-VAD-fmk. The ROS scavenger NAC efficiently suppressed not only ROS generation, but also caspase-3-mediated PARP cleavage, apoptosis, and release of cytochrome c and AIF, indicating a role of ROS in combined Dox + DMPS treatment-induced apoptotic death signaling. Taken together, these observations suggest that DMPS may be used as a therapeutic agent for overcoming drug-resistance in cancer cells by enhancing drug-induced apoptosis.

DGDA, a local sequence of the kringle 2 domain, is a functional motif of the tissue-type plasminogen activator's antiangiogenic kringle domain.

Antiangiogenic activity can be elicited by the kringle domains 1 and 2 of tissue-type plasminogen activator (TK1-2), or the kringle 2 domain alone. In a previous report, we showed that the anti-migratory effect of TK1-2 is mediated in part by its interference with integrin alpha2beta1. Since integrin alpha2beta1 interacts with collagen type I through the DGEA (Asp-Gly-Glu-Ala) amino acid sequence, and a similar sequence, DGDA (Asp-Gly-Asp-Ala), exists in the kringle 2 domain, we investigated whether the DGDA sequence has a role in antiangiogenic activity of TK1-2. In an adhesion assay, the DGDA peptide inhibited adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized TK1-2. Pretreatment of the DGDA peptide also blocked anti-migratory activity of TK1-2. When the DGDA peptide alone was tested for antiangiogenic activity, it effectively inhibited VEGF-induced migration of HUVECs and tube formation on Matrigel. In addition, the DGDA peptide decreased differentiation of endothelial progenitor cells on collagen type I matrix. These data suggest that the DGDA sequence presents a functional epitope of TK1-2 and that it can be used as a potential novel antiangiogenic peptide.

Tumor angiogenesis promoted by ex vivo differentiated endothelial progenitor cells is effectively inhibited by an angiogenesis inhibitor, TK1-2.

Neovascularization plays a critical role in the growth and metastatic spread of tumors and involves recruitment of circulating endothelial progenitor cells (EPC) from bone marrow as well as sprouting of preexisting endothelial cells. In this study, we examined if EPCs could promote tumor angiogenesis and would be an effective cellular target for an angiogenesis inhibitor, the recombinant kringle domain of tissue-type plasminogen activator (TK1-2). When TK1-2 was applied in the ex vivo culture of EPCs isolated from human cord blood, TK1-2 inhibited adhesive differentiation of mononuclear EPCs into endothelial-like cells. In addition, it inhibited the migration of ex vivo cultivated EPCs and also inhibited their adhesion to fibronectin matrix or endothelial cell monolayer. When A549 cancer cells were coimplanted along with ex vivo cultivated EPCs s.c. in nude mice, the tumor growth was increased. However, the tumor growth and the vascular density of tumor tissues increased by coimplanted EPCs were decreased upon TK1-2 treatment. Accordingly, TK1-2 treatment reduced the remaining number of EPCs in tumor tissues and their incorporation into the host vascular channels. In addition, overall expression levels of vascular endothelial growth factor (VEGF) and von Willebrand factor in tumor tissues were decreased upon TK1-2 treatment. Interestingly, strong VEGF expression by implanted EPCs was decreased by TK1-2. Finally, we confirmed in vitro that TK1-2 inhibited VEGF secretion of EPCs. TK1-2 also inhibited endothelial cell proliferation and migration induced by the conditioned medium of EPCs. Therefore, we concluded that EPCs, as well as mature endothelial cells, could be an important target of TK1-2.

Antimigratory effect of TK1-2 is mediated in part by interfering with integrin alpha2beta1.

The recombinant two kringle domain of human tissue-type plasminogen activator (TK1-2) has been shown to inhibit endothelial cell proliferation, angiogenesis, and tumor cell growth despite of sharing a low amino acid sequence homology with angiostatin. Here, we explored a possible inhibitory mechanism of action of TK1-2 by focusing on antimigratory effect. TK1-2 effectively inhibited endothelial cell migration induced by basic fibroblast growth factor or vascular endothelial growth factor in a dose-dependent manner and tube formation on Matrigel. It blocked basic fibroblast growth factor-induced or vascular endothelial growth factor-induced phosphorylation of extracellular signal-regulated kinase 1/2 and formation of actin stress fibers and focal adhesions. Interestingly, TK1-2 alone induced the weak phosphorylation of focal adhesion kinase, whereas it inhibited focal adhesion kinase phosphorylation induced by growth factors. When immobilized, TK1-2 promoted adhesion and spreading of endothelial cells compared with bovine serum albumin. However, treatment with anti-alpha(2)beta(1) blocking antibody markedly diminished endothelial cell adhesion to immobilized TK1-2 compared with anti-alpha(v)beta(3) or anti-alpha(5)beta(1) antibody. Pretreatment of soluble TK1-2 also altered the binding level of anti-alpha(2)beta(1) antibody to endothelial cells in fluorescence-activated cell sorting analysis. Indeed, a blocking antibody against integrin alpha(2)beta(1) or knocking down of integrin alpha(2) expression prevented the inhibitory effect of TK1-2 in cell migration. Therefore, these results suggest that TK1-2 inhibits endothelial cell migration through inhibition of signaling and cytoskeleton rearrangement in part by interfering with integrin alpha(2)beta(1).

Autophagy Is a Potential Target for Enhancing the Anti-Angiogenic Effect of Mebendazole in Endothelial Cells.

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has recently been noted as a repositioning candidate for angiogenesis inhibition and cancer therapy. However, the definite anti-angiogenic mechanism of MBZ remains unclear. In this study, we explored the inhibitory mechanism of MBZ in endothelial cells (ECs) and developed a novel strategy to improve its anti-angiogenic therapy. Treatment of ECs with MBZ led to inhibition of EC proliferation in a dose-dependent manner in several culture conditions in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or FBS, without selectivity of growth factors, although MBZ is known to inhibit VEGF receptor 2 kinase. Furthermore, MBZ inhibited EC migration and tube formation induced by either VEGF or bFGF. However, unexpectedly, treatment of MBZ did not affect FAK and ERK1/2 phosphorylation induced by these factors. Treatment with MBZ induced shrinking of ECs and caused G2-M arrest and apoptosis with an increased Sub-G1 fraction. In addition, increased levels of nuclear fragmentation, p53 expression, and active form of caspase 3 were observed. The marked induction of autophagy by MBZ was also noted. Interestingly, inhibition of autophagy through knocking down of Beclin1 or ATG5/7, or treatment with autophagy inhibitors such as 3-methyladenine and chloroquine resulted in marked enhancement of anti-proliferative and pro-apoptotic effects of MBZ in ECs. Consequently, we suggest that MBZ induces autophagy in ECs and that protective autophagy can be a novel target for enhancing the anti-angiogenic efficacy of MBZ in cancer treatment.

Integrin alphavbeta3 is not significantly implicated in the anti-migratory effect of anti-angiogenic urokinase kringle domain.

The recombinant kringle domain (UK1) of urokinase-type plasminogen activator (uPA) has been shown to possess anti-angiogenic activity in vitro and in vivo. It has also been found to inhibit in vivo malignant glioma growth. In contrast, direct interaction of the kringle domain of uPA and integrin alphavbeta3 has been reported to be involved in plasminogen and leukocyte activation by uPA. Since integrin alphavbeta3 is involved in tumor angiogenesis, we investigated whether integrin alphavbeta3 is involved in the inhibitory function of UK1 in angiogenesis, by examining its anti-migratory activity. In a modified Boyden chamber assay, the Pichia-expressed UK1 dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells (HUVECs). However, in the absence of growth factor stimulation, soluble UK1 alone did not induce or inhibit HUVEC migration. In cell adhesion, immobilized UK1 promoted HUVEC adhesion and spreading which were compared to BSA. Pretreatment of the anti-alphavbeta3 integrin antibody, significantly inhibited HUVEC binding to immobilized UK1, whereas neither anti-alpha2beta1 nor anti-alpha5beta1 integrin antibody had any effect, although pre-treatment of the soluble UK1 showed no marked alteration of the binding level of anti-alphavbeta3 antibody to HUVECs in FACS analysis. In a modified Boyden chamber assay, the function blocking antibodies against integrins alphavbeta3, alpha2beta1 and alpha5beta1 did not completely prevent the inhibitory effect of UK1 in HUVEC migration. These results suggest that UK1 interacts with integrin alphavbeta3, but its anti-migratory activity on endothelial cells is not significantly mediated by integrin alphavbeta3.

The kringle domain of tissue-type plasminogen activator inhibits extracellular matrix-induced adhesion and migration of endothelial cells.

The recombinant two-kringle domain of human tissue-type plasminogen activator (TK1-2) was found to inhibit angiogenesis and tumor growth. Recently, we found that TK1-2 inhibits adhesive differentiation of endothelial progenitor cells, and its contribution to tumor angiogenesis. In this study, we investigated the effects of TK1-2 on extracellular matrix-induced adhesion, signaling, and migration in order to understand the mechanism of action of TK1-2. When human umbilical vein endothelial cells were pretreated with TK1-2 and then allowed to adhere to immobilized fibronectin, vitronectin, or gelatin, cell adhesion to all the tested matrices decreased dose-dependently upon TK1-2 treatment. TK1-2 also inhibited the formation of actin stress fibers and focal adhesions upon attachment to each matrix. Moreover, fibronectin- and vitronectin-induced endothelial cell migration was dose-dependently inhibited by TK1-2. TK1-2 also suppressed fibronectin-induced ERK1/2 phosphorylation. Hence the results suggest that disturbance of extracellular matrix-induced adhesion, signaling, and migration of endothelial cells is involved in the anti-angiogenic activity of TK1-2.

Urokinase-derived peptide UP-7 suppresses tumor angiogenesis and metastasis through inhibition of FAK activation.

The recombinant kringle domain of urokinase (UK1) has been shown to inhibit angiogenesis and brain tumor growth in vivo. To avoid limitations in application due to mass production of recombinant protein, we attempted to develop a novel peptide inhibitor from UK1 sequence consisting of 83 amino acids that contains α-helices, loops and ß-sheets. We dissected UK1 sequence to seven peptides based on structure and amino acid characteristics, and examined the anti-angiogenic activities for the constructed peptides. Among the tested peptides, UP-7 most potently inhibited the proliferation and migration of endothelial cells (ECs) in vitro, and also potently inhibited in vivo angiogenesis in the mouse matrigel plug assay. Such anti-angiogenic activities were not exerted by the scrambled peptide. At molecular level, UP-7 inhibited growth factor-induced phosphorylation of FAK and ERK1/2. It also suppressed formation of stress fibers and focal adhesions and also inhibited the attachment and spreading of ECs onto immobilized fibronectin. In a lung cancer animal model xenografted with non-UP-7-sensitive NCI-H460 cells, systemic treatment of UP-7 effectively suppressed tumor growth through inhibition of angiogenesis. Interestingly, breast cancer cells such as LM-MDA-MB-231 cells were moderately sensitive to UP-7 in proliferation differently from other cancer cells. UP-7 also inhibited migration, invasion and FAK phosphorylation of LM-MDA-MB-231 cells. Accordingly, UP-7 potently inhibited lung metastatic growth of LM-MDA-MB-231 cells in an experimental metastasis model. Taken together, these results suggest that novel peptide UP-7 can be effectively used for treatment of breast cancer metastatic growth through inhibition of angiogenesis and invasion.

Outgrowing endothelial progenitor-derived cells display high sensitivity to angiogenesis modulators and delayed senescence.

Outgrowing endothelial progenitor-derived cells (EPDCs) originate from a novel hierarchy of endothelial progenitor cells. In this study, EPDCs isolated from human cord blood were examined for phenotype and functional features upon aging. Young or aged EPDCs were similar to human umbilical vein endothelial cells (HUVECs), in exhibiting typical endothelial phenotypes. However, EPDCs were more sensitive to angiogenesis inducers or inhibitors in proliferation and migration. In addition, EPDCs underwent senescence markedly slowly with sustained endothelial NO synthase expression and activation, and their ability to undergo capillary morphogenesis was retained throughout longterm culture. Thus, these results suggest that a homogenous population of EPDCs derived from clonogenic expansion may provide an effective vasculogenesis tool.