Perifascial areolar tissue graft promotes angiogenesis and wound healing in an exposed ischemic component rabbit model.

Multiple studies have reported the use of perifascial areolar tissue (PAT) grafts to treat wounds involving exposed ischemic tissues, avascular structures, and defective membrane structures. Our objective was to assess the quantitative effects of PAT grafts and their suitability for wounds with ischemic tissue exposure and to qualitatively determine the factors through which PAT promotes wound healing and repair. We conducted histological, immunohistochemical, and mass spectrometric analyses of the PAT grafts. PAT grafts contain numerous CD34+ progenitor/stem cells, extracellular matrix, growth factors, and cytokines that promote wound healing and angiogenesis. Furthermore, we established a male rabbit model to compare the efficacy of PAT grafting with that of an occlusive dressing treatment (control) for wounds with cartilage exposure. PAT grafts could cover ischemic components with granulation tissue and promote angiogenesis. Macroscopic and histological observations of the PAT graft on postoperative day seven revealed capillaries bridging the ischemic tissue (vascular bridging). Additionally, the PAT graft suppressed wound contraction and alpha smooth muscle actin (αSMA) levels and promoted epithelialization. These findings suggested that PAT can serve as a platform to enhance wound healing and promote angiogenesis. This is the first study to quantify the therapeutic efficacy of PAT grafts, revealing their high value for the treatment of wounds involving exposed ischemic structures. The effectiveness of PAT grafts can be attributed to two primary factors: vascular bridging and the provision of three essential elements (progenitor/stem cells, extracellular matrix molecules, and growth factors/cytokines). Moreover, PAT grafts may be used as transplant materials to mitigate excessive wound contraction and the development of hypertrophic scarring.

Transthyretin Is Commonly Upregulated in the Hippocampus of Two Stress-Induced Depression Mouse Models.

Chronic stress can affect gene expression in the hippocampus, which alters neural and cerebrovascular functions, thereby contributing to the development of mental disorders such as depression. Although several differentially expressed genes in the depressed brain have been reported, gene expression changes in the stressed brain remain underexplored. Therefore, this study examines hippocampal gene expression in two mouse models of depression induced by forced swim stress (FSS) and repeated social defeat stress (R-SDS). Transthyretin (Ttr) was commonly upregulated in the hippocampus of both mouse models, as determined by microarray, RT-qPCR, and Western blot analyses. Evaluation of the effects of overexpressed Ttr in the hippocampus using adeno-associated virus-mediated gene transfer revealed that TTR overexpression induced depression-like behavior and upregulation of Lcn2 and several proinflammatory genes (Icam1 and Vcam1) in the hippocampus. Upregulation of these inflammation-related genes was confirmed in the hippocampus obtained from mice vulnerable to R-SDS. These results suggest that chronic stress upregulates Ttr expression in the hippocampus and that Ttr upregulation may be involved in the induction of depression-like behavior.

EPAC2 acts as a negative regulator in Matrigel-driven tubulogenesis of human microvascular endothelial cells.

Angiogenesis is physiologically essential for embryogenesis and development and reinitiated in adult animals during tissue growth and repair. Forming new vessels from the walls of existing vessels occurs as a multistep process coordinated by sprouting, branching, and a new lumenized network formation. However, little is known regarding the molecular mechanisms that form new tubular structures, especially molecules regulating the proper network density of newly formed capillaries. This study conducted microarray analyses in human primary microvascular endothelial cells (HMVECs) plated on Matrigel. The RAPGEF4 gene that encodes exchange proteins directly activated by cAMP 2 (EPAC2) proteins was increased in Matrigel-driven tubulogenesis. Tube formation was suppressed by the overexpression of EPAC2 and enhanced by EPAC2 knockdown in endothelial cells. Endothelial cell morphology was changed to round cell morphology by EPAC2 overexpression, while EPAC2 knockdown showed an elongated cell shape with filopodia-like protrusions. Furthermore, increased EPAC2 inhibited endothelial cell migration, and ablation of EPAC2 inversely enhanced cell mobility. These results suggest that EPAC2 affects the morphology and migration of microvascular endothelial cells and is involved in the termination and proper network formation of vascular tubes.

Sp6/Epiprofin is a master regulator in the developing tooth.

Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study, whole-genome ChIP-seq analysis was applied to identify the transcriptional regulatory gene targets of Sp6 in mesenchymal cells of the developing tooth. Bioinformatic analysis of a pool of Sp6 target peaks identified the consensus nine nucleotide binding DNA motif CTg/aTAATTA. Consistent with these findings, a number of enamel and dentin matrix genes including amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam) and dental sialophosphoprotein (Dspp), were identified to contain Sp6 target sequences. Sp6 peaks were also found in other important tooth genes including transcription factors (Dlx2, Dlx3, Dlx4, Dlx5, Sp6, Sp7, Pitx2, and Msx2) and extracellular matrix-related proteins (Col1a2, Col11a2, Halpn1). Unsupervised UMAP clustering of tooth single cell RNA-seq data confirmed the presence of Sp6 transcripts co-expressed with many of the identified target genes within ameloblasts and odontoblasts. Lastly, transcriptional reporter assays using promoter fragments from the Hapln1 and Sp6 gene itself revealed that Sp6 co-expression enhanced gene transcriptional activity. Taken together these results highlight that Sp6 is a major regulator of multiple extracellular matrix genes in the developing tooth.

Protective Effects of Collagen Tripeptides in Human Aortic Endothelial Cells by Restoring ROS-Induced Transcriptional Repression.

Collagen tripeptide (CTP) is defined as a functional food material derived from collagenase digests of type I collagen and contains a high concentration of tripeptides with a Gly-X-Y sequence. CTP has several biological effects, including the acceleration of fracture healing, ameliorating osteoarthritis, and improving dryness and photoaging of the skin. Recently, an antiatherosclerotic effect of CTP has been reported, although its molecular mechanism is yet to be determined. In this study, we examined the effects of CTP on primary cultured human aortic endothelial cells (HAECs) under oxidative stress, because oxidative endothelial dysfunction is a trigger of atherosclerosis. DNA microarray and RT-qPCR analyses showed that CTP treatment recovered the downregulated expression of several genes, including the interleukin-3 receptor subunit alpha (IL3RA), which were suppressed by reactive oxygen species (ROS) treatment in HAECs. Furthermore, IL3RA knockdown significantly decreased the viability of HAECs compared with control cells. RT-qPCR analysis also showed that solute carrier 15 family peptide transporters, which are involved in CTP absorption into cells, were expressed in HAECs at levels more than comparable to those of a CTP-responsive human osteoblastic cell line. These results indicated that CTP exerts a protective effect for HAECs, at least in part, by regulating the recovery of ROS-induced transcriptional repression.

Emerging Role of AP-1 Transcription Factor JunB in Angiogenesis and Vascular Development.

Blood vessels are essential for the formation and maintenance of almost all functional tissues. They play fundamental roles in the supply of oxygen and nutrition, as well as development and morphogenesis. Vascular endothelial cells are the main factor in blood vessel formation. Recently, research findings showed heterogeneity in vascular endothelial cells in different tissue/organs. Endothelial cells alter their gene expressions depending on their cell fate or angiogenic states of vascular development in normal and pathological processes. Studies on gene regulation in endothelial cells demonstrated that the activator protein 1 (AP-1) transcription factors are implicated in angiogenesis and vascular development. In particular, it has been revealed that JunB (a member of the AP-1 transcription factor family) is transiently induced in endothelial cells at the angiogenic frontier and controls them on tip cells specification during vascular development. Moreover, JunB plays a role in tissue-specific vascular maturation processes during neurovascular interaction in mouse embryonic skin and retina vasculatures. Thus, JunB appears to be a new angiogenic factor that induces endothelial cell migration and sprouting particularly in neurovascular interaction during vascular development. In this review, we discuss the recently identified role of JunB in endothelial cells and blood vessel formation.

Role of Decorin in Posterior Capsule Opacification and Eye Lens Development.

Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)ß-2 showed a decrease in DCN expression. The expression of tropomyosin 1 (Tpm1), a marker of lens EMT increased after the addition of TGFß-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mice for lens-specific hDCN (hDCN-Tg). Injury-induced EMT of the mouse lens, and the expression patterns of α smooth muscle actin, were attenuated in hDCN-Tg mice lenses. Overexpression of DCN inhibited the TGFß-2-induced upregulation of Tpm1 and EMT observed during wound healing of the lens, but it did not affect mouse lens morphology until 48 weeks of age. Our findings demonstrate that DCN plays a significant role in regulating EMT formation of LECs and PCO, and suggest that for therapeutic intervention, maintenance of physiological expression of DCN is essential to attenuate EMT progression and PCO formation.

Metformin Mitigates DPP-4 Inhibitor-Induced Breast Cancer Metastasis via Suppression of mTOR Signaling.

The biological influence of antidiabetic drugs on cancer cells and diabetic cancer patients has not yet been completely elucidated. We reported that a dipeptidyl peptidase (DPP)-4 inhibitor accelerates mammary cancer metastasis by inducing epithelial-mesenchymal transition (EMT) through the CXCL12/CXCR4/mTOR axis. Metformin has been shown to inhibit the mTOR signaling pathway. In this study, we investigated whether metformin mitigates breast cancer metastasis induced by a DPP-4 inhibitor via suppression of mTOR signaling. In cultured mouse mammary and human breast cancer cells, metformin suppressed DPP-4 inhibitor KR62436 (KR)-induced EMT and cell migration via suppression of the mTOR pathway associated with AMPK activation. For the in vivo study, metformin intervention was performed in an allograft 4T1 breast cancer model mouse with or without KR. We also analyzed mice transplanted with shRNA-mediated DPP-4 knockdown 4T1 cells. Treatment with metformin inhibited the lung metastasis of DPP-4-deficient 4T1 mammary tumor cells generated by either KR administration or DPP-4 knockdown. Immunostaining of primary tumors indicated that DPP-4 suppression promoted the expression of EMT-inducing transcription factor Snail through activation of the CXCR4-mediated mTOR/p70S6K pathway in an allograft breast cancer model; metformin abolished this alteration. Metformin treatment did not alter DPP-4-deficiency-induced expression of CXCL12 in either plasma or primary tumors. Our findings suggest that metformin may serve as an antimetastatic agent by mitigating the undesirable effects of DPP-4 inhibitors in patients with certain cancers. IMPLICATIONS: Metformin could combat the detrimental effects of DPP-4 inhibitor on breast cancer metastasis via mTOR suppression, suggesting the potential clinical relevance. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/1/61/F1.large.jpg.

Role of Arginine Methylation in Alternative Polyadenylation of VEGFR-1 (Flt-1) pre-mRNA.

Mature mRNA is generated by the 3' end cleavage and polyadenylation of its precursor pre-mRNA. Eukaryotic genes frequently have multiple polyadenylation sites, resulting in mRNA isoforms with different 3'-UTR lengths that often encode different C-terminal amino acid sequences. It is well-known that this form of post-transcriptional modification, termed alternative polyadenylation, can affect mRNA stability, localization, translation, and nuclear export. We focus on the alternative polyadenylation of pre-mRNA for vascular endothelial growth factor receptor-1 (VEGFR-1), the receptor for VEGF. VEGFR-1 is a transmembrane protein with a tyrosine kinase in the intracellular region. Secreted forms of VEGFR-1 (sVEGFR-1) are also produced from the same gene by alternative polyadenylation, and sVEGFR-1 has a function opposite to that of VEGFR-1 because it acts as a decoy receptor for VEGF. However, the mechanism that regulates the production of sVEGFR-1 by alternative polyadenylation remains poorly understood. In this review, we introduce and discuss the mechanism of alternative polyadenylation of VEGFR-1 mediated by protein arginine methylation.

Ultraviolet B-induced Otx2 expression in lens epithelial cells promotes epithelial-mesenchymal transition.

Ultraviolet (UV) radiation of eyes is a major risk factor for cataractogenesis, although the molecular mechanisms underlying this process remain poorly understood and genes that are affected by UV radiation have not been fully identified. In this study, we examined the UV-related gene regulation in lens epithelial cells (LECs) of mouse eyes and investigated the molecular mechanisms of UV-triggered cataractogenesis. Forty-one genes were significantly upregulated in LECs following UVB exposure in vivo in two independent experiments. Among these, Otx2 was strongly upregulated in LECs, suggesting that it may act as an upstream regulator of UVB-induced changes in gene expression. Accordingly, Otx2 overexpression in LECs in vitro induced morphological changes in cell shapes. Epithelial-mesenchymal transition (EMT)-related molecules, such as TGFß2, αSMA and fibronectin were upregulated in Otx2-overexpressing LECs, concomitant with suppression of lens fiber cell marker genes, such as CRYAA and DNASEIIB. In vitro experiments suggested that UVB upregulated Otx2 through hydrogen peroxide generation. Aberrant upregulation of Otx2 in LECs following UV irradiation induces the EMT and alteration of the lens cell characteristics, likely contributing to cataractogenesis.

Inhibition of Dipeptidyl Peptidase-4 Accelerates Epithelial-Mesenchymal Transition and Breast Cancer Metastasis via the CXCL12/CXCR4/mTOR Axis.

Dipeptidyl peptidase (DPP)-4 is a multifunctional glycoprotein involved in various biological and pathologic processes. DPP-4 has been widely recognized as a therapeutic target for type 2 diabetes mellitus but is also implicated in the development of human malignancies. Here, we show that inhibition of DPP-4 accelerates breast cancer metastasis via induction of CXCL12/CXCR4, which activates mTOR to promote epithelial-mesenchymal transition (EMT). In cultured cells, DPP-4 knockdown induced EMT and cell migration. Treatment with the DPP-4 inhibitor KR62436 (KR) promoted primary tumor growth and lung metastasis in a 4T1 tumor allograft mouse model; DPP-4 knockdown in 4T1 cells displayed similar phenotypes in vivo and in vitro. KR treatment enhanced the levels of CXCL12/CXCR4 and phosphorylated mTOR, which were associated with the induction of EMT in metastatic cancer cells. KR-induced EMT in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 or the mTOR inhibitor rapamycin, and AMD3100 suppressed KR-induced metastasis in vivo. Our findings suggest that DPP-4 plays a significant role in cancer biology and that inhibition of DPP-4 promotes cancer metastasis via induction of the CXCL12/CXCR4/mTOR/EMT axis. SIGNIFICANCE: These findings reveal that inhibition of DPP-4 increases the metastatic potential of breast cancer. This is especially important given the potential use of DPP-4 inhibition as a therapeutic strategy for type 2 diabetes.

JunB regulates angiogenesis and neurovascular parallel alignment in mouse embryonic skin.

Blood vessels and nerve fibers are often closely arranged in parallel throughout the body. Therefore, neurovascular interactions have been suggested to be important for the development of vascular networks. However, the molecular mechanisms and genes regulating this process remain unclear. In the present study, we investigated the genes that are activated in endothelial cells (ECs) following interactions with neurons during vascular development. Microarray analyses of human primary microvascular ECs co-cultured with mouse primary dorsal root ganglion cells showed that JunB is strongly upregulated in ECs by neurovascular interactions. Furthermore, the forced expression of JunB in ECs stimulated a tip-like cell formation and angiogenesis in vitro and induced vascular endothelial growth factor A (VEGFA) and the pro-angiogenic integrin subunit ITGB3 expression. Moreover, in vivo knockdown of JunB in ECs from developing mouse limb skin considerably decreased the parallel alignments of blood vessels and nerve fibers. Taken together, the present data demonstrates for the first time that JunB plays an important role in the formation of embryonic vascular networks. These results contribute to the molecular understanding of neurovascular interactions during embryonic vascular development.

Regulation of soluble Flt-1 (VEGFR-1) production by hnRNP D and protein arginine methylation.

Soluble fms-like tyrosine kinase-1 (sFlt-1) functions as a potent inhibitor of angiogenesis by trapping vascular endothelial growth factor (VEGF). However, the precise regulatory mechanism of sFlt-1 production is unknown. Here, we report that vascular sFlt-1 production is regulated by heterogeneous nuclear ribonucleoprotein D (hnRNP D) and arginine methylation. We showed that hnRNP D bound to Flt-1 pre-mRNA and that hnRNP D overexpression decreased sFlt-1 mRNA in human microvascular endothelial cells (HMVECs). In contrast, the reduction of hnRNP D levels induced an increase in sFlt-1 production. Overexpression of an hnRNP D mutant in which the arginine residue of the known arginine methylation motif (arginine-glycine-glycine; RGG) was replaced with alanine did not reduce the level of soluble-form RNA produced from the Flt-1 minigene. Moreover, we demonstrated that overexpression of arginine methyltransferase decreased the soluble-form RNA level, whereas overexpression of arginine demethylase and addition of methyltransferase inhibitors increased sFlt-1 mRNA levels. These findings indicate that hnRNP D and arginine methylation play important roles in the regulation of Flt-1 mRNA alternative polyadenylation.

Osteocytic cell necrosis is caused by a combination of glucocorticoid-induced Dickkopf-1 and hypoxia.

Osteonecrosis is a major glucocorticoid-induced complication in the orthopedics field. Despite the extensive researches, mechanisms underlining the glucocorticoid-induced osteonecrosis are largely unknown. Here, we first provide the evidence that a combined treatment of cultured osteocytic cells with glucocorticoid and hypoxia caused necrotic cell death, which is assumed to occur in the acute bone injuries induced by glucocorticoids. We cultured MLO-Y4 murine osteocytic cells under hypoxia in the presence or absence of Dexamethasone (Dex) and examined the rates of apoptotic and necrotic cell death. Dex or hypoxia alone increased apoptotic cells, but not necrotic cells. The combination of Dex and hypoxia dramatically increased osteocytic cell death, notably necrotic cell death. The expression of Dickkopf-1 (Dkk-1), an inhibitor of Wnt/ß-catenin signal, was scarcely expressed in the control and hypoxic cells, but a dramatic increase of the Dkk-1 expression was detected in Dex-treated cells. siRNA-mediated knockdown of Dkk-1 in Dex and hypoxia-treated osteocytic cells showed the significant decreases in both apoptotic and necrotic cells. The results indicated that the combination of Dkk-1 overexpression by Dex and hypoxia causes the necrotic osteocytic cell death. The results also indicated that blocking of Dkk-1 can protect bone cells from glucocorticoid and hypoxia-induced cell injury.

Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation.

The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development.

Hypoxia down-regulates sFlt-1 (sVEGFR-1) expression in human microvascular endothelial cells by a mechanism involving mRNA alternative processing.

sFlt-1 (soluble Flt-1) potently inhibits angiogenesis by binding extracellularly to VEGF (vascular endothelial growth factor). In the present paper, we report that hypoxia down-regulates sFlt-1 expression in HMVECs (human microvascular endothelial cells), a constituent of microvessels where angiogenesis occurs. Hypoxia (5-1% O2) increased VEGF expression in HMVECs. In contrast, the levels of sFlt-1 mRNA and protein in HMVECs decreased significantly as the O2 concentration fell, whereas mFlt-1 (membrane-bound Flt-1) mRNA and protein remained unchanged. This suggested that hypoxia selectively regulates alternative 3'-end processing of sFlt-1 pre-mRNA. We have also demonstrated that sFlt-1 overexpression in lentiviral-construct-infected HMVECs counteracted VEGF-induced endothelial cell growth. We next identified cis-elements involved in sFlt-1 mRNA processing in HMVECs using a human Flt-1 minigene and found that two non-contiguous AUUAAA sequences function as the poly(A) signal. Furthermore, we identified a cis-element in intron 13 that regulates sFlt-1 mRNA processing. Mutagenesis of the U-rich region in intron 13 caused a significant decrease in the soluble-form/membrane-form RNA ratio in the minigene-transfected HMVECs. These results suggest that decreased sFlt-1 expression due to hypoxia contributes to hypoxia-induced angiogenesis and reveals a novel mechanism regulating angiogenesis by alternative mRNA 3'-end processing.

Role of Epiprofin, a zinc-finger transcription factor, in limb development.

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. In the present work, we have investigated the role of Epiprofin (Epfn/Sp6), a member of the SP/KLF transcription factor family that is expressed in the limb ectoderm and the AER, during limb development. Epfn mutant mice have a defective autopod that shows mesoaxial syndactyly in the forelimb and synostosis (bony fusion) in the hindlimb and partial bidorsal digital tips. Epfn mutants also show a defect in the maturation of the AER that appears flat and broad, with a double ridge phenotype. By genetic analysis, we also show that Epfn is controlled by WNT/b-CATENIN signaling in the limb ectoderm. Since the less severe phenotypes of the conditional removal of b-catenin in the limb ectoderm strongly resemble the limb phenotype of Epfn mutants, we propose that EPFN very likely functions as a modulator of WNT signaling in the limb ectoderm.

Matrix metalloproteinase-9 associated with heparan sulphate chains of GPI-anchored cell surface proteoglycans mediates motility of murine colon adenocarcinoma cells.

Using murine colon adenocarcinoma-derived clones with different metastatic potentials, the cellular localization of matrix metalloporteinase-9 (MMP-9) and its role in the cell motility were examined. Highly metastatic LuM1 clone aggressively invaded into adjacent tissue in vivo, but low metastatic NM11 clone did not. As compared with the NM11 clone, the LuM1 clone expressed and secreted a remarkably large amount of MMP-9, and exhibited higher abilities of cell migration and invasion in vitro, which were suppressed by MMP-2/MMP-9 inhibitor IV. MMP-9, exhibiting high affinity to heparin, was demonstrated to be condensed on tips of cellular podia. Treatment of the cells with heparitinase-I or heparin resulted in release of MMP-9 from the cell surface, which caused concomitant suppression of their motility to a similar level to that with the MMP inhibitor. Immunoprecipitation of a LuM1 cell lysate with an anti-MMP-9 antibody resulted in co-precipitation of phosphatidylinositol-specific phospholipase C-susceptible heparan sulphate proteoglycans having 66 and 64 kDa core proteins. Taken together, the present results demonstrate that secreted MMP-9 associates with glypican-like proteoglycans through their heparan sulphate chains, and plays a crucial role in cell motility of LuM1 cells.

A novel function of syndecan-2, suppression of matrix metalloproteinase-2 activation, which causes suppression of metastasis.

The syndecans comprise a family of cell surface heparan sulfate proteoglycans exhibiting complex biological functions involving the interaction of heparan sulfate side chains with a variety of soluble and insoluble heparin-binding extracellular ligands. Here we demonstrate an inverse correlation between the expression level of syndecan-2 and the metastatic potential of three clones derived from Lewis lung carcinoma 3LL. This correlation was proved to be a causal relationship, because transfection of syndecan-2 into the higher metastatic clone resulted in the suppression of both spontaneous and experimental metastases to the lung. Although the expression levels of matrix metalloproteinase-2 (MMP-2) and its cell surface activators, such as membrane-type 1 matrix metalloproteinase and tissue inhibitor of metalloproteinase-2, were similar regardless of the metastatic potentials of the clones, elevated activation of MMP-2 was observed in the higher metastatic clone. Removal of heparan sulfate from the cell surface of low metastatic cells by treatment with heparitinase-I promoted MMP-2 activation, and transfection of syndecan-2 into highly metastatic cells suppressed MMP-2 activation. Furthermore, transfection of mutated syndecan-2 lacking glycosaminoglycan attachment sites into highly metastatic cells did not have any suppressive effect on MMP-2 activation, suggesting that this suppression was mediated by the heparan sulfate side chains of syndecan-2. Actually, MMP-2 was found to exhibit a strong binding ability to heparin, the dissociation constant value being 62 nM. These results indicate a novel function of syndecan-2, which acts as a suppressor for MMP-2 activation, causing suppression of metastasis in at least the metastatic system used in the present study.

Inhibition of experimental lung metastases of Lewis lung carcinoma cells by chemically modified heparin with reduced anticoagulant activity.

Heparin, a widely used anticoagulant, is known to have anti-metastatic activity, although the mechanism is not fully understood. In the present study, we investigated the mechanism of this anti-metastatic activity using periodate-oxidized and borohydride-reduced heparin with low anticoagulant activity (LAC heparin). The anticoagulant activity of LAC heparin is markedly reduced to almost the control level in terms of prothrombin time in vitro, and no hemorrhagic complication was observed with injection of LAC heparin into mice in vivo. LAC heparin injected intravenously with Lewis lung carcinoma cells or 10 min before tumor cell injection significantly inhibited, to the same extent as intact heparin and in a dose- and time-dependent manner, the lung colonization that develops after intravenous injection (i.v.) of tumor cells. Flow cytometric analysis revealed that Lewis lung carcinoma cells strongly express heparan sulfate on their surface. Both the LAC heparin and intact heparin inhibited the adhesion and invasion of tumor cells to Matrigel-coated dishes in vitro without significant effect on the tumor cell growth. LAC heparin also significantly diminished tumor cell retention in the lung after i.v. of LacZ gene-tagged Lewis lung carcinoma cells. These results suggest that LAC heparin may prevent tumor cells from attachment to the subendothelial matrix of lung capillaries by competitively inhibiting cell surface heparan sulfate functions and suppress lung colonization.