Cdon deficiency causes cardiac remodeling through hyperactivation of WNT/ß-catenin signaling.

On pathological stress, Wnt signaling is reactivated and induces genes associated with cardiac remodeling and fibrosis. We have previously shown that a cell surface receptor Cdon (cell-adhesion associated, oncogene regulated) suppresses Wnt signaling to promote neuronal differentiation however its role in heart is unknown. Here, we demonstrate a critical role of Cdon in cardiac function and remodeling. Cdon is expressed and predominantly localized at intercalated disk in both mouse and human hearts. Cdon-deficient mice develop cardiac dysfunction including reduced ejection fraction and ECG abnormalities. Cdon-/- hearts exhibit increased fibrosis and up-regulation of genes associated with cardiac remodeling and fibrosis. Electrical remodeling was demonstrated by up-regulation and mislocalization of the gap junction protein, Connexin 43 (Cx43) in Cdon-/- hearts. In agreement with altered Cx43 expression, functional analysis both using Cdon-/- cardiomyocytes and shRNA-mediated knockdown in rat cardiomyocytes shows aberrant gap junction activities. Analysis of the underlying mechanism reveals that Cdon-/- hearts exhibit hyperactive Wnt signaling as evident by ß-catenin accumulation and Axin2 up-regulation. On the other hand, the treatment of rat cardiomyocytes with a Wnt activator TWS119 reduces Cdon levels and aberrant Cx43 activities, similarly to Cdon-deficient cardiomyocytes, suggesting a negative feedback between Cdon and Wnt signaling. Finally, inhibition of Wnt/ß-catenin signaling by XAV939, IWP2 or dickkopf (DKK)1 prevented Cdon depletion-induced up-regulation of collagen 1a and Cx43. Taken together, these results demonstrate that Cdon deficiency causes hyperactive Wnt signaling leading to aberrant intercellular coupling and cardiac fibrosis. Cdon exhibits great potential as a target for the treatment of cardiac fibrosis and cardiomyopathy.

MRI Monitoring of Tumor-Selective Anticancer Drug Delivery with Stable Thermosensitive Liposomes Triggered by High-Intensity Focused Ultrasound.

Monitoring of drug release from a heat-activated liposome carrier provides an opportunity for real-time control of drug delivery and allows prediction of the therapeutic effect. We have developed short-chain elastin-like polypeptide-incorporating thermosensitive liposomes (STLs). Here, we report the development of STL encapsulating gadobenate dimeglumine (Gd-BOPTA), a MRI contrast agent, and doxorubicin (Dox) (Gd-Dox-STL). The Dox release profile from Gd-Dox-STL was comparable to Gd-Dox-LTSL; however, the serum stability of Gd-Dox-STL was much higher than Gd-Dox-LTSL. MRI studies showed that the difference in T1 relaxation time between 37 and 42 °C for Gd-Dox-STL was larger than the difference for Gd-Dox-LTSL. Although relaxivity for both liposomes at 42 °C was similar, the relaxivity of Gd-Dox-STL at 37 °C was 2.5-fold lower than that of Gd-Dox-LTSL. This was likely due to Gd-BOPTA leakage from the LTSL because of low stability at 37 °C. Pharmacokinetic studies showed plasma half-lives of 4.85 and 1.95 h for Gd-Dox-STL and Gd-Dox-LTSL, respectively, consistent with in vitro stability data. In vivo MRI experiments demonstrated corelease of Dox and Gd-BOPTA from STL under mild hyperthermia induced by high-intensity focused ultrasound (HIFU), which suggests STL is a promising tumor selective formulation when coupled with MR-guided HIFU.

Preventive effects of CTLA4Ig-overexpressing adipose tissue--derived mesenchymal stromal cells in rheumatoid arthritis.

BACKGROUND AIMS: Rheumatoid arthritis is a systemic autoimmune disorder. In this study, we first compared the therapeutic effects of syngeneic and xenogeneic adipose tissue-derived stem cells on a collagen-induced arthritis mouse model. Second, we investigated the synergistic preventive effects of CTLA4Ig and adipose tissue-derived mesenchymal stromal cells (ASCs) as a therapeutic substance. METHODS: Arthritis was induced in all groups except for the normal, saline (N) group, using chicken type II collagen (CII). Animals were divided into C (control, saline), H (hASCs), M (mASCs) and N groups (experiment I) and C, H, CT (CTLA4Ig-overexpressing human ASC [CTLA4Ig-hASCs]) and N groups (experiment II), according to transplanted material. Approximately 2 × 10(6) ASCs or 150 µL of saline was intravenously administered on days 24, 27, 30 and 34, and all animals were killed on days 42 to 44 after CII immunization. RESULTS: Anti-mouse CII autoantibodies were significantly lower in the H, M and CT groups than in the C group. Cartilage damage severity score and C-telopeptide of type II collagen were significantly lower in the CT group than in the C group. The serum levels of IL-6 were significantly lower in the H, M and CT groups than in the C group. The serum levels of keratinocyte chemoattractant were significantly lower in the CT group than the C group. CONCLUSIONS: There were similar effects of ASCs on the decrease of anti-mouse CII autoantibody levels between syngeneic and xenogeneic transplantations, and CTLA4Ig-hASCs showed synergistic preventive effects compared with non-transduced hASCs.

Endothelial progenitor cell homing: prominent role of the IGF2-IGF2R-PLCbeta2 axis.

Homing of endothelial progenitor cells (EPCs) to the neovascular zone is now considered to be an essential step in the formation of vascular networks during embryonic development and also for neovascularization in postnatal life. We report here the prominent role of the insulin-like growth factor 2 (IGF2)/IGF2 receptor (IGF2R) system in promoting EPC homing. With high-level expression of IGF2R in EPCs, IGF2-induced hypoxic conditions stimulated multiple steps of EPC homing in vitro and promoted both EPC recruitment and incorporation into the neovascular area, resulting in enhanced angiogenesis in vivo. Remarkably, all IGF2 actions were exerted predominantly through IGF2R-linked G(i) protein signaling and required intracellular Ca(2+) mobilization induced by the beta2 isoform of phospholipase C. Together, these findings indicate that locally generated IGF2 at either ischemic or tumor sites may contribute to postnatal vasculogenesis by augmenting the recruitment of EPCs. The utilization of the IGF2/IGF2R system may therefore be useful for the development of novel means to treat angiogenesis-dependent diseases.

Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-kappaB activation in endothelial cells.

Visfatin has recently been identified as a novel visceral adipokine which may be involved in obesity-related vascular disorders. However, it is not known whether visfatin directly contributes to endothelial dysfunction. Here, we investigated the effect of visfatin on vascular inflammation, a key step in a variety of vascular diseases. Visfatin induced leukocyte adhesion to endothelial cells and the aortic endothelium by induction of the cell adhesion molecules, ICAM-1 and VCAM-1. Promoter analysis revealed that visfatin-mediated induction of CAMs is mainly regulated by nuclear factor-kappaB (NF-kappaB). Visfatin stimulated IkappaBalpha phosphorylation, nuclear translocation of the p65 subunit of NF-kappaB, and NF-kappaB DNA binding activity in HMECs. Furthermore, visfatin increased ROS generation, and visfatin-induced CAMs expression and NF-kappaB activation were abrogated in the presence of the direct scavenger of ROS. Taken together, our results demonstrate that visfatin is a vascular inflammatory molecule that increases expression of the inflammatory CAMs, ICAM-1 and VCAM-1, through ROS-dependent NF-kappaB activation in endothelial cells.

COMP-angiopoietin-1 decreases lipopolysaccharide-induced acute kidney injury.

During sepsis endothelial dysfunction is an important pathogenetic mechanism in acute kidney injury (AKI). Lipopolysaccharide (LPS)-induced endotoxemia is associated with renal hemodynamic changes such as alterations of renal blood flow (RBF), vascular resistance, and glomerular filtration rate. We used adenoviral delivery of an engineered variant of native angiopoietin-1 (COMP-angiopoietin-1) containing anti-inflammatory and anti-permeability functions, to determine if regulation of renal endothelial cell dysfunction may have a beneficial role in preventing AKI during LPS-induced endotoxemia in mice. This treatment prevented the endotoxin-induced decrease of RBF and mean arterial pressure while improving glomerular filtration rate. Treatment also mitigated the effects of LPS on renal intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 protein expression, the number of ER-HR3-positive macrophages that infiltrated the kidney, serum nitrate/nitrite levels, renal inducible nitric oxide synthase protein expression, the induction of tubular epithelial reactive oxygen and nitrogen species, and renal microvascular permeability. Our findings show that COMP-angiopoietin-1, an endothelium-oriented therapeutic agent, protects against AKI caused by endotoxemia.

Colloidal gold nanoparticles as a blood-pool contrast agent for X-ray computed tomography in mice.

OBJECTIVES: To present the pharmacokinetics and computed tomographic imaging efficacy of colloidal gold nanoparticles (AuNPs) as a blood-pool agent for x-ray computed tomography (CT). METHODS AND MATERIALS: To prepare the colloidal AuNPs, gold nanocrystals were modified using sulfhydrated polyethylene glycol (PEG). Cytotoxicity and histopathologic tests were carried out for toxicity evaluation. Six adult Balb/c mice underwent microcomputed tomography scans after injection of colloidal AuNPs (2.5 micromol Au/g body weight). Four mice with HT-1080 tumors were imaged for visualization of the tumor vasculature. RESULTS: The PEG coated colloidal AuNPs appeared as spherical nanoparticles with 38-nm diameters. The AuNPs-PEG showed a biocompatibility without toxicity in the mice. We identified a stable imaging window for visualizing the vasculature system, immediately to 24 hours after injection. Microcomputed tomography imaging using AuNPs-PEG clearly visualized the tumor vascular structures. CONCLUSION: Colloidal AuNPs show potential as a blood-pool agent for x-ray CT imaging.

Cloning and characterization of zebrafish CTCF: Developmental expression patterns, regulation of the promoter region, and evolutionary aspects of gene organization.

CTCF is a nuclear phosphoprotein capable of using different subsets of its 11 Zn fingers (ZF) for sequence-specific binding to many dissimilar DNA CTCF-target sites. Such sites were identified in the genomic DNA of various multicellular organisms, in which the CTCF gene was cloned, including insects, birds, rodents, and primates. CTCF/DNA-complexes formed in vivo with different 50-bp-long sequences mediate diverse functions such as positive and negative regulation of promoters, and organization of all known enhancer-blocking elements ("chromatin insulators") including constitutive and epigenetically regulated elements. Abnormal functions of certain CTCF sites are implicated in cancer and in epigenetic syndromes such as BWS and skewed X-inactivation. We describe here the cloning and characterization of the CTCF cDNA and promoter region from zebrafish, a valuable vertebrate model organism. The full-length zebrafish CTCF cDNA clone is 4244 bp in length with an open reading frame (ORF) of 2391 bp that encodes 797 amino acids. The zebrafish CTCF amino acid sequence shows high identity (up to 98% in the zinc finger region) with human CTCF, and perfect conservation of exon-intron organization. Southern blot analyses indicated that the zebrafish genome contains a single copy of the CTCF gene. In situ hybridization revealed the presence of zebrafish CTCF transcripts in all early stages of embryogenesis. Transfection assays with luciferase reporter-constructs identified a core promoter region within 146 bp immediately upstream of the transcriptional start site of zebrafish CTCF that is located at a highly conserved YY1/Initiator element.

Gliotoxin reduces the severity of trinitrobenzene sulfonic acid-induced colitis in mice: evidence of the connection between heme oxygenase-1 and the nuclear factor-kappaB pathway in vitro and in vivo.

BACKGROUND: Gliotoxin, a fungal metabolite, has been known to show strong immunosuppressive properties, although its mechanisms are not completely understood. In this report, the authors investigated the mechanism whereby gliotoxin has anti-inflammatory properties in vitro and in trinitrobenzene sulfonic acid-induced colitis. MATERIALS AND METHODS: Body weight, histological scores, and myeloperoxidase activity were evaluated in trinitrobenzene sulfonic acid colitis. Nuclear factor-kappaB (NF-kappaB) p65, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-12, and intercellular adhesion molecule-1 were detected by immunohistochemical staining. IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Heme oxygenase-1 (HO-1) expression and I-kappaB degradation were analyzed by Western blot. RESULTS: Pretreatment of human epithelial HT-29 cells with gliotoxin significantly blocked the I-kappaB degradation and NF-kappaB p65 nuclear translocation induced by tumor necrosis factor-alpha or IL-1beta; these were parallel with the inhibition of IL-8 secretion and intercellular adhesion molecule-1 expression in the same cells. Interestingly, gliotoxin induced HO-1 in HT-29 cells and, in turn, inhibition of HO-1 activity by a zinc protoporphyrin IX reversed the effects of gliotoxin in terms of I-kappaB degradation, intercellular adhesion molecule-1 expression, and IL-8 production. In trinitrobenzene sulfonic acid colitis, gliotoxin administration significantly improved the clinical and histopathological symptoms. Notably, gliotoxin also induced HO-1 in the colonic mucosa and zinc protoporphyrin IX reversed the protective effects of gliotoxin in trinitrobenzene sulfonic acid colitis. CONCLUSIONS: These results demonstrate for the first time that the anti-inflammatory actions mediated by gliotoxin include HO-1 induction and the subsequent blockade of NF-kappaB-dependent signaling pathways in vitro and in vivo. The current results also demonstrate that gliotoxin may be an effective agent for the treatment of diseases characterized by mucosal inflammation.

Compact soft x-ray transmission microscopy with sub-50 nm spatial resolution.

In this paper, the development of compact transmission soft x-ray microscopy (XM) with sub-50 nm spatial resolution for biomedical applications is described. The compact transmission soft x-ray microscope operates at lambda = 2.88 nm (430 eV) and is based on a tabletop regenerative x-ray source in combination with a tandem ellipsoidal condenser mirror for sample illumination, an objective micro zone plate and a thinned back-illuminated charge coupled device to record an x-ray image. The new, compact x-ray microscope system requires the fabrication of proper x-ray optical devices in order to obtain high-quality images. For an application-oriented microscope, the alignment procedure is fully automated via computer control through a graphic user interface. In imaging studies using our compact XM system, a gold mesh image was obtained with 45 nm resolution at x580 magnification and 1 min exposure. Images of a biological sample (Coscinodiscus oculoides) were recorded.

Neuromedin B receptor antagonism inhibits migration, invasion, and epithelial-mesenchymal transition of breast cancer cells.

Neuromedin B (NMB) acts as an autocrine growth factor and a pro-angiogenic factor. Its receptor, NMB receptor (NMB-R), is overexpressed in solid tumors. In the present study, we showed that an NMB-R antagonist, PD168368, suppresses migration and invasion of the human breast cancer cell line MDA-MB-231. In addition, PD168368 reduced epithelial-mesenchymal transition (EMT) of breast cancer cells by E-cadherin upregulation and vimentin downregulation. Moreover, we found that PD168368 potently inhibits in vivo metastasis of breast cancer. Taken together, these findings suggest that NMB-R antagonism may be an alternative approach to prevent breast cancer metastasis, and targeting NMB-R may provide a novel therapeutic strategy for breast cancer treatment.

Prmt7 Deficiency Causes Reduced Skeletal Muscle Oxidative Metabolism and Age-Related Obesity.

Maintenance of skeletal muscle function is critical for metabolic health and the disruption of which exacerbates many chronic diseases such as obesity and diabetes. Skeletal muscle responds to exercise or metabolic demands by a fiber-type switch regulated by signaling-transcription networks that remains to be fully defined. Here, we report that protein arginine methyltransferase 7 (Prmt7) is a key regulator for skeletal muscle oxidative metabolism. Prmt7 is expressed at the highest levels in skeletal muscle and decreased in skeletal muscles with age or obesity. Prmt7(-/-) muscles exhibit decreased oxidative metabolism with decreased expression of genes involved in muscle oxidative metabolism, including PGC-1α. Consistently, Prmt7(-/-) mice exhibited significantly reduced endurance exercise capacities. Furthermore, Prmt7(-/-) mice exhibit decreased energy expenditure, which might contribute to the exacerbated age-related obesity of Prmt7(-/-) mice. Similarly to Prmt7(-/-) muscles, Prmt7 depletion in myoblasts also reduces PGC-1α expression and PGC-1α-promoter driven reporter activities. Prmt7 regulates PGC-1α expression through interaction with and activation of p38 mitogen-activated protein kinase (p38MAPK), which in turn activates ATF2, an upstream transcriptional activator for PGC-1α. Taken together, Prmt7 is a novel regulator for muscle oxidative metabolism via activation of p38MAPK/ATF2/PGC-1α.

Effects of Transplantation of CTLA4Ig-Overexpressing Adipose Tissue-Derived Mesenchymal Stem Cells in Mice With Sustained Severe Rheumatoid Arthritis.

CTLA4Ig has therapeutic potential for rheumatoid arthritis patients unresponsive to methotrexate (MTX) or TNF-α blockers. However, recombinant CTLA4Ig proteins are short acting and expensive. Adipose tissue-derived mesenchymal stem cells (ASCs) present an ideal stem cell source for practical regenerative medicine due to their abundant availability and their beneficial properties including immunomodulation, homing activity, paracrine effects, and differentiation ability. Therefore, we aimed to determine whether CTLA4Ig and human ASCs show synergistic effects on immunomodulation and clinical improvement of sustained severe rheumatoid arthritis in a mouse model. hASCs overexpressing CTLA4Ig (CTLA4Ig-hASC) were serially transplanted into mice with collagen-induced arthritis. Arthritic mice were subjected to four treatments based on their arthritis score on day 62 postimmunization: control (C group), hASC (H group), CTLA4Ig-hASC (CT group), and MTX (MTX group). A group of healthy mice was used as a normal control (N). Mice in the N and C groups were infused with 150 µl saline, and 2 × 10(6) hASCs or CTLA4Ig-hASCs in 150 µl of saline were intravenously administered to those in the H and CT groups, respectively, on days 63, 70, 77, and 84 after CII immunization. About 1 mg/kg of methotrexate was intraperitoneally administered to the MTX group three times a week for 4 weeks. Serial hASC and CTLA4Ig-hASC transplantation modulated various cytokines and chemokines related to the development of rheumatoid arthritis. Both treatments protected against destruction of cartilage, with CTLA4Ig-hASCs being most effective. Serum levels of CII autoantibodies and C-telopeptide of type II collagen were significantly low in the group transplanted with CTLA4Ig-hASCs. In vitro, ASC and CTLA4Ig-hASC treatment significantly decreased T-bet and GATA-3 expression in splenocytes from arthritic mice, and CTLA4Ig-hASC treatment significantly increased the ratio of Treg/Th17 (CD4(+)CD25(+)FoxP3(+)/CD4(+)CD25(+)RORγt) cells. Serial hASC and CTLA4Ig-hASC transplantation offers promising treatment for rheumatoid arthritis, and CTLA4Ig-hASCs showed stronger therapeutic effects than nontransduced hASCs.

Exploitation of astrocytes by glioma cells to facilitate invasiveness: a mechanism involving matrix metalloproteinase-2 and the urokinase-type plasminogen activator-plasmin cascade.

The presence of reactive astrocytes around glioma cells in the CNS suggests the possibility that these two cell types could be interacting. We addressed whether glioma cells use the astrocyte environment to modulate matrix metalloproteinase-2 (MMP-2), a proteolytic enzyme implicated in the invasiveness of glioma cells. We found that astrocytes in culture produce significant amounts of the pro-form of MMP-2 but undetectable levels of active MMP-2. However, after coculture with the U251N glioma line, astrocyte pro-MMP-2 was converted to the active form. The mechanism of pro-MMP-2 activation in glioma-astrocyte coculture was investigated and was found to involve the urokinase-type plasminogen activator (uPA)-plasmin cascade whereby uPA bound to uPA receptor (uPAR), leading to the conversion of plasminogen to plasmin. The latter cleaved pro-MMP-2 to generate its active form. Furthermore, key components (i.e., uPAR, uPA, and pro-MMP-2) were contributed principally by astrocytes, whereas the U251N glioma cells provided plasminogen. In correspondence with this biochemical cascade, the transmigration of U251N cells through Boyden invasion chambers coated with an extracellular matrix barrier was increased significantly in the presence of astrocytes, and this was inhibited by agents that disrupted the uPA-plasmin cascade. Finally, using resected human glioblastoma specimens, we found that tumor cells, but not astrocytes, expressed plasminogen in situ. We conclude that glioma cells exploit their astrocyte environment to activate MMP-2 and that this leads to the increased invasiveness of glioma cells.

Hypoxia-responsive element-mediated soluble Tie2 vector exhibits an anti-angiogenic activity in vitro under hypoxic condition.

Hypoxia-inducible factor-1 (HIF-1) is one of the key mammalian transcription factors and shows increased levels in both protein stability and intrinsic transcriptional activity during low oxygen tension. Hypoxia-activated functional HIF-1 protein binds to hypoxia-responsive elements (HRE) in the enhancers of several genes including VEGF, the major player in angiogenesis, and initiates their mRNA expression. The molecular mechanisms regulating the gene expression under hypoxic conditions could increase the therapeutic window of tumor-specific delivery systems. In this study, to examine hypoxia-specific production of anti-angiogenic therapeutic gene, we constructed 5 copies of HRE (5xHRE) of human VEGF linked to soluble Tie2 (sTie2) driven by minimal SV40 promoter (5xHRE/SV40/sTie2). Our data showed that under hypoxia the secreted sTie2 selectively inhibited tube formation and migration capacities of endothelial cells in vitro. Hence, we propose that the vector system, 5xHRE/SV40/sTie2, might be a useful tool for down-regulating tumor angiogenesis under hypoxic condition.

p11 regulates extracellular plasmin production and invasiveness of HT1080 fibrosarcoma cells.

The defining characteristic of a tumor cell is its ability to escape the constraints imposed by neighboring cells, invade the surrounding tissue, and metastasize to distant sites. This invasive property of tumor cells is dependent on activation of proteases at the cell surface. Most cancer cells secrete the urokinase-type plasminogen activator, which converts cell-bound plasminogen to plasmin. Here we address the issue of whether the plasminogen binding protein, p11, plays a significant role in this process. Transfection of human HT1080 fibrosarcoma cells with the human p11 gene in the antisense orientation resulted in a loss of p11 protein from the cell surface and concomitant decreases in cellular plasmin production, ECM degradation, and cellular invasiveness. The transfected cells demonstrated reduced development of lung metastatic foci in SCID mice. In contrast, HT1080 cells transfected with the p11 gene in the sense orientation displayed increased cell surface p11 protein and concomitant increases in cellular plasmin production, as well as enhanced ECM degradation and enhanced cellular invasiveness. The p11 overexpressing cells showed enhanced development of lung metastatic foci. These data establish that changes in the extracellular expression of the plasminogen receptor protein, p11, dramatically affect tumor cell-mediated pericellular proteolysis.

Transplantation of Adipose Tissue-Derived Mesenchymal Stem Cells Prevents the Development of Lupus Dermatitis.

MRL/lpr mice spontaneously develop high titers of anti-dsDNA antibodies and symptoms such as glomerular nephritis and organ weight gain. They also develop spontaneous skin inflammation similar to the cutaneous lesions common in human lupus erythematosus. This study aimed to compare the effects of long-term serial administration of human adipose tissue-derived mesenchymal stem cells (ASCs), CTLA4Ig-overexpressing ASCs, and cyclophosphamide treatment in MRL/lpr mice. MRL/lpr mice were divided into saline (C), cyclophosphamide (Y), ASC early (E), ASC late (L), and CTLA4Ig-overexpressing ASC (CT) treatment groups. Background-matched control MRL/MPJ mice treated with saline (N) were also compared. The treatment period was 5-23 weeks, except for the L group (15-23 weeks). Blood and tissue samples were collected when the mice were 24 weeks old. Organ weight, anti-dsDNA antibodies, urine protein, skin and kidney histologic abnormalities, and trabecular bone volume were evaluated. The Y group showed the greatest decrease in anti-dsDNA antibodies, organ weight, degree of kidney inflammation and glomerular infiltration of C3, and incidence rate of severe proteinuria; the E, L, and CT treatment groups showed better results than the C group. ASC transplantation reduced anti-dsDNA antibody levels significantly. Mice treated with ASCs or CTLA4Ig-ASCs starting from the early disease stage did not show dermatitis upon gross examination; they demonstrated significant improvement in hyperkeratosis, acanthosis, and inflammatory cell infiltration scores in histopathology. Micro-CT analysis revealed that cyclophosphamide treatment significantly decreased bone volume and increased bone spacing in the trabecular bone. Thus, we found that ASC and CTLA4-ASC treatments prevent lupus dermatitis development in MRL/lpr mice without adverse effects.

Insulin-like growth factor-II regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line HaCaT.

Psoriasis is a chronic, relapsing skin disease characterized by enhanced angiogenesis. The pathogenetic process resulting in hypervascularity remains to be further investigated. It has been reported that a potent angiogenic factor, vascular endothelial growth factor (VEGF) is overexpressed in psoriatic epidermis and that the level of insulin-like growth factor II (IGF-II) is significantly elevated in the tissue fluid and serum of the psoriatic lesion. We considered the possibility that IGF-II might function as a paracrine inducer of VEGF. Here, we demonstrated that exposure of HaCaT keratinocytes to IGF-II induced both mRNA and protein expression of VEGF through the MAP kinase (extracellular signal-regulated kinase (ERK2) pathway. Particularly, we determined that phosphorylation of ERK2 but not p38 and JNK1/2 was activated by IGF-II in a time-dependent manner. Additionally, we found that IGF-II treatment induced the expression of MDM2 through the MAP kinase pathway. Moreover, the increase of MDM2 resulted in decreased levels of p53 followed by increased expression of HIF-1alpha and VEGF. Taken together, these results suggest that IGF-II enhances the expression of VEGF in HaCaT cells by increasing HIF-1alpha levels.

Hypoxia-induced angiogenesis during carcinogenesis.

The formation of new blood vessels, angiogenesis, is an essential process during development and disease. Angiogenesis is well known as a crucial step in tumor growth and progression. Angiogenesis is induced by hypoxic conditions and regulated by the hypoxia-inducible factor 1 (HIF-1). The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of the major HIF-1 target gene, vascular endothelial cell growth factor (VEGF). In this review, a brief overview of the mechanism of angiogenesis is discussed, focusing on the regulatory processes of the HIF-1 transcription factor. HIF-1 consists of a constitutively expressed HIF-1 beta (HIF-1beta) subunit and an oxygen-regulated HIF-1 alpha (HIF-1a) subunit. The stability and activity of HIF-1alpha are regulated by the interaction with various proteins, such as pVHL, p53, and p300/CBP as well as by post-translational modifications, hydroxylation, acetylation, and phosphorylation. It was recently reported that HIF-1alpha binds a co-activator of the AP-1 transcription factor, Jab-1, which inhibits the p53-dependent degradation of HIF-1 and enhances the transcriptional activity of HIF-1 and the subsequent VEGF expression under hypoxic conditions. ARD1 acetylates HIF-1alpha and stimulates pVHL-mediated ubiquitination of HIF-1alpha. With a growing knowledge of the molecular mechanisms in this field, novel strategies to prevent tumor angiogenesis can be developed, and from these, new anticancer therapies may arise.

Cooperation of endothelial and smooth muscle cells derived from human induced pluripotent stem cells enhances neovascularization in dermal wounds.

Human induced pluripotent stem cells (hiPSCs) are generated through the reprogramming of somatic cells into an embryonic stem cell-like state, such that vascular cells differentiated from hiPSCs might be a suitable autologous cell source for vascular regeneration. The goal of this study was to assess whether cotransplantation of endothelial cells (ECs) and smooth muscle cells (SMCs) differentiated from hiPSCs could promote neovascularization and tissue repair in a murine dermal wound model. hiPSCs were differentiated into ECs and SMCs; the differentiated cells displayed cell-specific surface markers. Compared to primary somatic cells, ECs and SMCs, which were differentiated from hiPSCs, strongly cooperated to enhance in vitro tubular network formation. In vivo gel assays in athymic nude mice showed that the coimplantation of differentiated ECs and SMCs significantly increased vascularization, unlike that observed in the case of implantation of differentiated ECs alone. In a murine full-thickness wound model, when compared with the transplantation of primary somatic cells or phosphate-buffered saline, cotransplantation of differentiated ECs and SMCs markedly enhanced neovascularization in injured tissues and accelerated wound healing. These results demonstrate that cotransplantation of hiPSC-derived ECs and SMCs may be feasible as a new autologous cell therapy for neovascularization and tissue repair.