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1.  VEGF Is Involved in the Increase of Dermal Microvascular Permeability Induced by Tryptase 
ISRN Dermatology  2012;2012:941465.
Tryptases are predominantly mast cell-specific serine proteases with pleiotropic biological activities and play a critical role in skin allergic reactions, which are manifested with rapid edema and increases of vascular permeability. The exact mechanisms of mast cell tryptase promoting vascular permeability, however, are unclear and, therefore, we investigated the effect and mechanism of tryptase or human mast cells (HMC-1) supernatant on the permeability of human dermal microvascular endothelial cells (HDMECs). Both tryptase and HMC-1 supernatant increased permeability of HDMECs significantly, which was resisted by tryptase inhibitor APC366 and partially reversed by anti-VEGF antibody and SU5614 (catalytic inhibitor of VEGFR). Furthermore, addition of tryptase to HDMECs caused a significant increase of mRNA and protein levels of VEGF and its receptors (Flt-1 and Flk-1) by Real-time RT-PCR and Western blot, respectively. These results strongly suggest an important role of VEGF on the permeability enhancement induced by tryptase, which may lead to novel means of controlling allergic reaction in skin.
PMCID: PMC3361195  PMID: 22666607
2.  Tryptase Promotes Atherosclerotic Plaque Haemorrhage in ApoE-/- Mice 
PLoS ONE  2013;8(4):e60960.
Tryptase, the most abundant mast cell (MC) granule protein, plays an important role in atherosclerosis plaque development. To test the hypothesis that tryptase participates directly in atherosclerosis plaque haemorrhage, the gene sequence and siRNA for tryptase were cloned into a lentivirus carrier and atherosclerosis plaque haemorrhage models in ApoE-/- mice were constructed. After a cuffing-cervical artery operation, the mice were randomly divided into 6 groups. Hematoxylin and eosin(HE) staining showed that the cervical artery plaque area was much larger in the tryptase overexpression group compared to the other groups, and there was greater artery stenosis. The artery stenosis from the cuff-side in all groups was more than 90%, except the siRNA group. Tryptase promotes plaque haemorrhage distinctively because 50% of the mice in the tryptase overexpression group had plaque haemorrhage, while only 10% in the siRNA group did. The immunohistochemistry of the cervical artery plaque showed that plasminogen activator inhibitor-1 (PAI-1) expression was the lowest while tissue plasminogen activator (tPA), CD31, CD34 and VEGF was the highest in the tryptase overexpression groups. This observation was completely contrary to what was observed in the siRNA group. Tryptase promoted bEnd.3 cell growth, migration and capillary-like tube formation, which suggests that tryptase can promote microvessel angiogenesis. PAI-1 expression was inhibited, while tPA expression was increased by tryptase in bEnd.3 cells. Our in vivo and in vitro studies suggest that trypase can promote atherosclerotic plaque haemorrhage by promoting angiogenesis and regulating the balance of PAI-1 and tPA. Thus, regulating tryptase expression in MCs may provide a potential target for atherosclerosis treatment.
PMCID: PMC3615996  PMID: 23573292
3.  Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP-Dependent Mechanism and Wound Angiogenesis 
Journal of Cellular Physiology  2014;230(2):356-365.
Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (β-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated β-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β-AR activation reduced pro-angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, β-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β-AR agonists could be promising anti-angiogenic modulators in skin. J. Cell. Physiol. 230: 356–365, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
PMCID: PMC4263239  PMID: 24986762
4.  Degranulating mast cells in fibrotic regions of human tumors and evidence that mast cell heparin interferes with the growth of tumor cells through a mechanism involving fibroblasts 
BMC Cancer  2005;5:121.
The purpose of this study was to test the hypothesis that mast cells that are present in fibrotic regions of cancer can suppress the growth of tumor cells through an indirect mechanism involving peri-tumoral fibroblasts.
We first immunostained a wide variety of human cancers for the presence of degranulated mast cells. In a subsequent series of controlled in vitro experiments, we then co-cultured UACC-812 human breast cancer cells with normal fibroblasts in the presence or absence of different combinations and doses of mast cell tryptase, mast cell heparin, a lysate of the human mast cell line HMC-1, and fibroblast growth factor-7 (FGF-7), a powerful, heparin-binding growth factor for breast epithelial cells.
Degranulating mast cells were localized predominantly in the fibrous tissue of every case of breast cancer, head and neck cancer, lung cancer, ovarian cancer, non-Hodgkin's lymphoma, and Hodgkin's disease that we examined. Mast cell tryptase and HMC-1 lysate had no significant effect on the clonogenic growth of cancer cells co-cultured with fibroblasts. By contrast, mast cell heparin at multiple doses significantly reduced the size and number of colonies of tumor cells co-cultured with fibroblasts, especially in the presence of FGF-7. Neither heparin nor FGF-7, individually or in combination, produced any significant effect on the clonogenic growth of breast cancer cells cultured without fibroblasts.
Degranulating mast cells are restricted to peri-tumoral fibrous tissue, and mast cell heparin is a powerful inhibitor of clonogenic growth of tumor cells co-cultured with fibroblasts. These results may help to explain the well-known ability of heparin to inhibit the growth of primary and metastatic tumors.
PMCID: PMC1242221  PMID: 16176582
5.  Level of Endothelial Cell Apoptosis Required for a Significant Decrease in Microvessel Density 
Experimental cell research  2007;313(16):3645-3657.
Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF165) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.
PMCID: PMC2080879  PMID: 17720154
6.  Association of Mast Cell-Derived VEGF and Proteases in Dengue Shock Syndrome 
Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls.
Methodology/Principal Findings
The study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9.
As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.
Author Summary
To clarify the involvement of mast cells in the development of severe Dengue diseases, plasma levels of mast cell-derived mediators, namely vascular endothelial cell growth factor (VEGF), tryptase, and chymase, were estimated in Dengue patients and control subjects in Vietnam. The levels of the mediators were significantly increased in Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS) patients compared with those of Dengue fever (DF) and control (febrile illness and healthy subjects) patients, and the soluble form of VEGF receptors (sVEGFR)-1 and -2 levels were significantly changed in the patients with severe disease. After 2–4 days of admission, the mediator levels had returned to similar levels as those of DF and control subjects. Furthermore, the levels of the Th17 cell-derived mast-cell activators IL-9 and -17 were increased in DHF and DSS. In-vitro production of VEGF in human mast cells was significantly enhanced in the presence of IL-9 when these cells were inoculated with Dengue virus in the presence of human Dengue virus-immune serum. As mast cells are an important source of VEGF, and tryptase and chymase are considered to be specific markers for mast cell activation, mast cells and mast cell-derived mediators might participate in the development of DHF/DSS.
PMCID: PMC3283553  PMID: 22363824
7.  Processing of human protryptase in mast cells involves cathepsins L, B and C1 
Human β-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized tetramer. β-Protryptase in solution can be directly processed to the mature enzyme by cathepsin (CTS) L and CTSB, and sequentially processed by autocatalysis at R−3 followed by CTSC proteolysis. However, it is uncertain which cathepsin is involved in protryptase processing inside human mast cells, because murine BMMC from CTSC-deficient mice convert protryptase (proMMCP-6) is to mature MMCP-6. This suggests that other proteases are important for processing human β-protryptase. In the current study, reduction of either CTSB or CTSL activity inside HMC-1 cells by shRNA silencing or cathepsin-specific pharmacologic inhibitors substantially reduced mature β-tryptase formation. Similar reductions of tryptase levels in primary skin-derived mast cells were observed with these pharmacologic inhibitors. In contrast, protryptase processing was minimally reduced by shRNA silencing of CTSC. A putative pharmacologic inhibitor of CTSC markedly reduced tryptase levels, suggesting an off-target effect. Skin mast cells contain substantially greater amounts of CTSL and CTSB than HMC-1 cells, the opposite being found for CTSC. Both CTSL and CTSB co-localize to the secretory granule compartment of skin mast cells. Thus, CTSL and CTSB are central to the processing of protryptase(s) in human mast cells, and are potential targets for attenuating production of mature tryptase in vivo.
PMCID: PMC3150348  PMID: 21742978
8.  Metronomic small molecule inhibitor of Bcl-2 (TW-37) is anti-angiogenic and potentiates the anti-tumor effect of ionizing radiation 
To investigate the effect of a metronomic (low dose, high frequency) small molecule inhibitor of Bcl-2 (TW-37) in combination with radiotherapy on microvascular endothelial cells in vitro and in tumor angiogenesis in vivo.
Methods and materials
Primary human dermal microvascular endothelial cells (HDMEC) were exposed to ionizing radiation and/or TW-37, and colony formation as well as capillary sprouting in 3-D collagen matrices, was evaluated. Xenografts vascularized with human blood vessels were engineered by co-transplantation of human squamous cell carcinoma cells (OSCC3) and HDMEC seeded in highly porous biodegradable scaffolds into the subcutaneous space of immunodeficient mice. Mice were treated with metronomic TW-37 and/or radiation, and tumor growth was evaluated.
Low dose TW-37 sensitized primary endothelial cells to radiation-induced inhibition of colony formation. Low dose TW-37 or radiation partially inhibited endothelial cell sprout formation, while in combination these therapies abrogated new sprouting. Combination of metronomic TW-37 and low dose radiation inhibited tumor growth and resulted in significant increase in time to failure as compared to controls, whereas single agents did not. Notably, histopathological analysis revealed that tumors treated with TW-37 (with or without radiation) are more differentiated and showed more cohesive invasive fronts, which is consistent with less aggressive phenotype.
These results demonstrate that metronomic TW-37 potentiates the anti-tumor effects of radiotherapy, and suggest that patients with head and neck cancer might benefit from the combination of small molecule inhibitor of Bcl-2 and radiation therapy.
PMCID: PMC2946486  PMID: 20675079
Developmental therapeutics; Radiotherapy; Head and Neck Cancer; Apoptosis; Neovascularization
9.  Mast cell tryptase is a mitogen for cultured fibroblasts. 
Journal of Clinical Investigation  1991;88(2):493-499.
Mast cells appear to promote fibroblast proliferation, presumably through secretion of growth factors, although the molecular mechanisms underlying this mitogenic potential have not been explained fully by known mast cell-derived mediators. We report here that tryptase, a trypsin-like serine proteinase of mast cell secretory granules, is a potent mitogen for fibroblasts in vitro. Nanomolar concentrations of dog tryptase strongly stimulate thymidine incorporation in Chinese hamster lung and Rat-1 fibroblasts and increase cell density in both subconfluent and confluent cultures of these cell lines. Tryptase-induced cell proliferation appears proteinase-specific, as this response is not mimicked by pancreatic trypsin or mast cell chymase. In addition, low levels of tryptase markedly potentiate DNA synthesis stimulated by epidermal growth factor, basic fibroblast growth factor, or insulin. Inhibitors of catalytic activity decrease the mitogenic capacity of tryptase, suggesting, though not proving, the participation of the catalytic site in cell activation by tryptase. Differences in Ca++ mobilization and sensitivity to pertussis toxin suggest that tryptase and thrombin activate distinct signal transduction pathways in fibroblasts. These data implicate mast cell tryptase as a potent, previously unrecognized fibroblast growth factor, and may provide a molecular link between mast cell activation and fibrosis.
PMCID: PMC295370  PMID: 1864960
10.  CD36-mediated activation of endothelial cell apoptosis by an N-terminal recombinant fragment of thrombospondin-2 inhibits breast cancer growth and metastasis in vivo 
Thus far the clinical benefits seen in breast cancer patients treated with drugs targeting the vascular endothelial growth factor (VEGF) pathway are only modest. Consequently, additional antiangiogenic approaches for treatment of breast cancer need to be investigated. Thrombospondin-2 (TSP-2) has been shown to inhibit tumor growth and angiogenesis with a greater potency than the related molecule TSP-1. The systemic effects of TSP-2 on tumor metastasis and the underlying molecular mechanisms of the antiangiogenic activity of TSP-2 have remained poorly understood. We generated a recombinant fusion protein consisting of the N-terminal region of TSP-2 and the IgG-Fc1 fragment (N-TSP2-Fc) and could demonstrate that the antiangiogenic activity of N-TSP2-Fc is dependent on the CD36 receptor. We found that N-TSP2-Fc inhibited VEGF-induced tube formation of human dermal microvascular endothelial cells (HDMEC) on matrigel in vitro and that concurrent incubation of anti-CD36 antibody with N-TSP2-Fc resulted in tube formation that was comparable to untreated control. N-TSP2-Fc potently induced apoptosis of HDMEC in vitro in a CD36-dependent manner. Moreover, we could demonstrate a CD36 receptor-mediated loss of mitochondrial membrane potential and activation of caspase-3 in HDMEC in vitro. Daily intraperitoneal injections of N-TSP2-Fc resulted in a significant inhibition of the growth of human MDA-MB-435 and MDA-MB-231 tumor cells grown in the mammary gland of immunodeficient nude mice and in reduced tumor vascularization. Finally, increased serum concentrations of N-TSP2-Fc significantly inhibited regional metastasis to lymph nodes and distant metastasis to lung as shown by quantitative real-time alu PCR. These results identify N-TSP2-Fc as a potent systemic inhibitor of tumor metastasis and provide strong evidence for an important role of the CD36 receptor in mediating the antiangiogenic activity of TSP-2.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-010-1085-7) contains supplementary material, which is available to authorized users.
PMCID: PMC3291836  PMID: 20714802
Breast cancer; Thrombospondin-2; CD36; Metastasis; Angiogenesis
11.  Endothelial Cells Enhance Tumor Cell Invasion through a Crosstalk Mediated by CXC Chemokine Signaling1 
Neoplasia (New York, N.Y.)  2008;10(2):131-139.
Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells.
PMCID: PMC2244688  PMID: 18283335
12.  Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C51 
Both mast cells and complement participate in innate and acquired immunity. The current study examines whether β-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of β-tryptase, and the β-tryptase-activating polyanion. The B12 mAb was used to stabilize β-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric β-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by β-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by β-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both β-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by β-tryptase in releasates of activated skin mast cells. Of further biologic interest, β-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest β-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of β-tryptase and complement anaphylatoxins are detected.
PMCID: PMC2645414  PMID: 18424754
13.  Involvement of MITF-A, an alternative isoform of mi transcription factor, on the expression of tryptase gene in human mast cells 
Experimental & Molecular Medicine  2010;42(5):366-375.
Mast cells play a central role in the initiation and development of allergic diseases through release of various mediators. Tryptase has been known to be a key mediator in mast cell-mediated inflammatory reactions. In the present study, we investigated whether the transcription of tryptase gene in human mast cells was induced by microphthalmia (mi)-associated transcription factor (MITF). We observed that the human CD34+ progenitor-derived cultured mast cells and human mast cell line HMC-1 expressed strongly the transcripts of tryptase-beta1 and MITF-A, which is a MITF alterative splicing isoform. The transcriptional activity of tryptase gene was specifically higher in HMC-1 cells compared to the tryptase-negative cells. Using mutant constructs of tryptase promoter, we observed that two E-box (CANNTG) motifs including between -817 to -715 and -421 to -202 are able to involve in the transactivation of tryptase gene by MITF-A. In addition, the binding of these motifs-containing oligonucleotides to MITF proteins was detectable by EMGA using the nuclear extracts of HMC-1 cells and anti-MITF mAb. The overexpression of MITF-A elevated tryptase production by HMC-1 cells, while the introduction of specific siRNA against MITF attenuated the expression and enzymatic activity of tryptase. These data suggest that MITF might play a role in regulating the transcription of tryptase gene in human mast cells.
PMCID: PMC2877250  PMID: 20513998
alternative splicing; gene expression regulation; mast cells; microphthalmia-associated transcription factor; tryptases
14.  Enzastaurin, an inhibitor of PKCβ, Enhances Antiangiogenic Effects and Cytotoxicity of Radiation against Endothelial Cells1,2 
Translational Oncology  2008;1(4):195-201.
PURPOSE: Angiogenesis plays an important role in pancreas cancer pathobiology. Pancreatic tumor cells secrete vascular endothelial growth factor (VEGF), activating endothelial cell protein kinase C beta (PKCβ) that phosphorylates GSK3β to suppress apoptosis and promote endothelial cell proliferation and microvessel formation. We used Enzastaurin (Enz) to test the hypothesis that inhibition of PKCβ results in radiosensitization of endothelial cells in culture and in vivo. MATERIALS/METHODS: We measured PKCβ phosphorylation, VEGF pathway signaling, colony formation, and capillary sprout formation in primary human dermal microvessel endothelial cells (HDMECs) after Enz or radiation (RT) treatment. Microvessel density and tumor volume of human pancreatic cancer xenografts in nude mice were measured after treatment with Enz, RT, or both. RESULTS: Enz inhibited PKCβ and radiosensitized HDMEC with an enhancement ratio of 1.31 ± 0.05. Enz combined with RT reduced HDMEC capillary sprouting to a greater extent than either agent alone. Enz prevented radiation-induced GSK3β phosphorylation of serine 9 while having no direct effect on VEGFR phosphorylation. Treatment of xenografts with Enz and radiation produced greater reductions in microvessel density than either treatment alone. The reduction in microvessel density corresponded with increased tumor growth delay. CONCLUSIONS: Enz-induced PKCβ inhibition radiosensitizes human endothelial cells and enhances the antiangiogenic effects of RT. The combination of Enz and RT reduced microvessel density and resulted in increased growth delay in pancreatic cancer xenografts, without increase in toxicity. These results provide the rationale for combining PKCβ inhibition with radiation and further investigating such regimens in pancreatic cancer.
PMCID: PMC2582168  PMID: 19043530
15.  Cysteinyl cathepsins and mast cell proteases in the pathogenesis and therapeutics of cardiovascular diseases 
Pharmacology & therapeutics  2011;131(3):338-350.
The initiation and progression of cardiovascular diseases involve extensive arterial wall matrix protein degradation. Proteases are essential to these pathological events. Recent discoveries suggest that proteases do more than catabolize matrix proteins. During the pathogenesis of atherosclerosis, abdominal aortic aneuryms, and associated complications, cysteinyl cathepsins and mast cell tryptases and chymases participate importantly in vascular cell apoptosis, foam cell formation, matrix protein gene expression, and pro-enzyme, latent cytokine, chemokine, and growth factor activation. Experimental animal disease models have been invaluable in examining each of these protease functions. Deficiency and pharmacological inhibition of cathepsins or mast cell proteases have allowed their in vivo evaluation in the setting of pathological conditions. Recent discoveries of highly selective and potent inhibitors of cathepsins, chymase, and tryptase, and their applications in vascular diseases in animal models and non-vascular diseases in human trials, have led to the hypothesis that selective inhibition of cathepsins, chymases, and tryptase will benefit patients suffering from cardiovascular diseases. This review highlights recent discoveries from in vitro cell-based studies to experimental animal cardiovascular disease models, from protease knockout mice to treatments with recently developed selective and potent protease inhibitors, and from patients with cathepsin-associated non-vascular diseases to those affected by cardiovascular complications.
PMCID: PMC3134138  PMID: 21605595
cathepsin; mast cell; chymase; tryptase; atherosclerosis; abdominal aortic aneurysm; protease inhibitor
16.  Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells 
The inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear.
Human dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls.
Cell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC.
The increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds.
PMCID: PMC3653709  PMID: 23514369
17.  Promiscuous Processing of Human α/β-Protryptases by Cathepsins L, B, and C 
Human α- and β-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. β-Protryptase is sequentially processed in vitro by autocatalysis at R−3 followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R−3Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. β-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q−3) and mutated β-protryptase (R−3Q) as well as wild-type β-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and β-protryptases from human mast cells to their mature enzymatically active products.
PMCID: PMC3339195  PMID: 21562164
18.  Synthesis, Storage, and Release of Vascular Endothelial Growth Factor/Vascular Permeability Factor (VEGF/VPF) by Human Mast Cells: Implications for the Biological Significance of VEGF206 
Molecular Biology of the Cell  1998;9(4):875-884.
Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.
PMCID: PMC25314  PMID: 9529385
19.  Targeting Mast Cells Tryptase in Tumor Microenvironment: A Potential Antiangiogenetic Strategy 
BioMed Research International  2014;2014:154702.
Angiogenesis is a complex process finely regulated by the balance between angiogenesis stimulators and inhibitors. As a result of proangiogenic factors overexpression, it plays a crucial role in cancer development. Although initially mast cells (MCs) role has been defined in hypersensitivity reactions and in immunity, it has been discovered that MCs have a crucial interplay on the regulatory function between inflammatory and tumor cells through the release of classical proangiogenic factors (e.g., vascular endothelial growth factor) and nonclassical proangiogenic mediators granule-associated (mainly tryptase). In fact, in several animal and human malignancies, MCs density is highly correlated with tumor angiogenesis. In particular, tryptase, an agonist of the proteinase-activated receptor-2 (PAR-2), represents one of the most powerful angiogenic mediators released by human MCs after c-Kit receptor activation. This protease, acting on PAR-2 by its proteolytic activity, has angiogenic activity stimulating both human vascular endothelial and tumor cell proliferation in paracrine manner, helping tumor cell invasion and metastasis. Based on literature data it is shown that tryptase may represent a promising target in cancer treatment due to its proangiogenic activity. Here we focused on molecular mechanisms of three tryptase inhibitors (gabexate mesylate, nafamostat mesylate, and tranilast) in order to consider their prospective role in cancer therapy.
PMCID: PMC4177740  PMID: 25295247
20.  Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice 
Cultured skin substitutes (CSS) consisting of autologous fibroblasts and keratinocytes combined with biopolymers are an adjunctive treatment for large excised burns. CSS containing two cell types are limited by anatomical deficiencies, including lack of a vascular plexus, leading to slower vascularization after grafting than split-thickness autograft. To address this limitation, CSS were prepared containing human keratinocytes, fibroblasts, and dermal microvascular endothelial cells (HDMEC) isolated from a single skin sample. After 16 days in culture, control CSS and CSS containing HDMEC (CSS+EC) were grafted to full-thickness wounds in athymic mice. In CSS+EC in vitro, HDMEC persisted in the dermal substitutes and formed multicellular aggregates. One wk after grafting, HDMEC in CSS+EC organized into multicellular structures, some containing lumens. By 4 wk after grafting, HDMEC were found in linear and circular organizations resembling vascular analogs associated with basement membrane deposition. In some cases, colocalization of HDMEC with mouse perivascular cells was observed. The results demonstrate HDMEC transplantation in a clinically relevant cultured skin model, persistence of HDMEC after grafting, and HDMEC organization into vascular analogs in vitro and in vivo. All cells were derived from the same donor tissue, indicating the feasibility of preparing CSS containing autologous HDMEC for grafting to patients.—Supp, D. M., Wilson-Landy, K., Boyce, S. T. Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice.
PMCID: PMC1820617  PMID: 12039861
cultured skin substitute; endothelial cell; angiogenesis; tissue engineering
21.  Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression 
PLoS ONE  2012;7(7):e40790.
Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.
PMCID: PMC3399855  PMID: 22815822
22.  Vixapatin (VP12), a C-Type Lectin-Protein from Vipera xantina palestinae Venom: Characterization as a Novel Anti-angiogenic Compound 
Toxins  2012;4(10):862-877.
A C-type lectin-like protein (CTL), originally identified as VP12 and lately named Vixapatin, was isolated and characterized from Israeli viper Vipera xantina palestinae snake venom. This CTL was characterized as a selective α2β1 integrin inhibitor with anti-melanoma metastatic activity. The major aim of the present study was to prove the possibility that this protein is also a potent novel anti-angiogenic compound. Using an adhesion assay, we demonstrated that Vixapatin selectively and potently inhibited the α2 mediated adhesion of K562 over-expressing cells, with IC50 of 3 nM. 3 nM Vixapatin blocked proliferation of human dermal microvascular endothelial cells (HDMEC); 25 nM inhibited collagen I induced migration of human fibrosarcoma HT-1080 cells; and 50 nM rat C6 glioma and human breast carcinoma MDA-MB-231 cells. 1 µM Vixapatin reduced HDMEC tube formation by 75% in a Matrigel assay. Furthermore, 1 µM Vixapatin decreased by 70% bFGF-induced physiological angiogenesis, and by 94% C6 glioma-induced pathological angiogenesis, in shell-less embryonic quail chorioallantoic membrane assay. Vixapatin’s ability to inhibit all steps of the angiogenesis process suggest that it is a novel pharmacological tool for studying α2β1 integrin mediated angiogenesis and a lead compound for the development of a novel anti-angiogenic/angiostatic/anti-cancer drug.
PMCID: PMC3496993  PMID: 23162702
C-type lectin protein; Vixapatin (VP12); α2β1; integrin; adhesion; migration; tube formation; Matrigel; CAM assay; angiogenesis
23.  Crosstalk between mast cells and pancreatic cancer cells contributes to pancreatic tumor progression 
To assess the clinical and pathological significance of mast cell infiltration in human pancreatic cancer and evaluate crosstalk between mast cells and cancer cells in vitro.
Experimental Design
Immunohistochemistry for tryptase was performed on 53 pancreatic cancer specimens. Mast cell counts were correlated with clinical variables and survival. Serum tryptase activity from cancer patients was compared to patients with benign pancreatic disease. In vitro, the effect of pancreatic cancer conditioned media on mast cell migration was assessed. The effect of conditioned media from the human mast cell line, LAD-2, on cancer and normal ductal cell proliferation was assessed by thymidine incorporation. Matrigel invasion assays were used to evaluate the effect of mast cell conditioned media on cancer cell invasion in the presence and absence of a matrix metalloproteinase inhibitor, GM6001.
Mast cell infiltration was significantly increased in pancreatic cancer compared to normal pancreatic tissue [11.4±6.7vs.2.0±1.4(p<0.001)]. Increased infiltrating mast cells correlated with higher grade tumors (p<0.0001) and worse survival. Patients with pancreatic cancer had elevated serum tryptase activity (p<0.05). In vitro, AsPC1 and PANC-1 cells induced mast cell migration. Mast cell conditioned media induced pancreatic cancer cell migration, proliferation and invasion but had no effect on normal ductal cells. Furthermore, the effect of mast cells on cancer cell invasion was in large part MMP-dependent.
Tumor infiltrating mast cells are associated with worse prognosis in pancreatic cancer. In vitro, the interaction between mast cells and pancreatic cancer cells promote tumor growth and invasion.
PMCID: PMC3122919  PMID: 20371681
Mast cells; Pancreatic Adenocarcinoma; Tryptase; Matrix Metalloproteinase
24.  Connective Tissue Growth Factor (CTGF/CCN2) mediates angiogenic effect of S1P in human dermal microvascular endothelial cells 
The primary objective of this study was to examine the potential interaction between sphingosine-1-phosphate (S1P), a pleiotropic lipid mediator, and CTGF/CCN2 a secreted multimodular protein, in the process of endothelial cell migration. The second objective was to determine whether C- and N-terminal domains of CTGF/CCN2 have specific function in cell migration.
Materials and Methods
Migration of human dermal microvascular endothelial cells (HDMECs) was examined in monolayer wound healing “scratch” assay, while capillary-like tube formation was examined in 3 dimensional collagen co-culture assays.
We observed that S1P stimulates HDMECs migration concomitant with upregulation of CTGF/CCN2 expression. Furthermore, the blockade of endogenous CTGF/CCN2 via siRNA abrogated S1P induced HDMECs migration and capillary-like tube formation. Full length CTGF induced cell migration and capillary-like tube formation with potency similar to that of S1P, while C-terminal domain of CTGF was slightly less effective. However; N-terminal domain had only a residual activity in inducing capillary-like tube formation.
This study revealed that CTGF/CCN2 is required for the S1P induced endothelial cell migration, which suggests that CTGF/CCN2 may be an important mediator of S1P induced physiological and pathological angiogenesis. Moreover, this study shows that the pro-migratory activity of CTGF/CCN2 is located in the C-terminal domain.
PMCID: PMC3058265  PMID: 21166920
HDMECs; S1P; CTGF/CCN2; N-terminus of CTGF; C-terminus of CTGF
25.  Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo 
The Journal of Experimental Medicine  1996;183(6):2681-2686.
Stem cell factor (SCF), also known as mast cell growth factor, kit ligand, and steel factor, is the ligand for the tyrosine kinase receptor (SCFR) that is encoded by the c-kit proto-oncogene. We analyzed the effects of recombinant human SCF (r-hSCF, 5-50 micrograms/kg/day, injected subcutaneously) on mast cells and melanocytes in a phase I study of 10 patients with advanced breast carcinoma. A wheal and flare reaction developed at each r-hSCF injection site; by electron microscopy, most dermal mast cells at these sites exhibited extensive, anaphylactic-type degranulation. A 14-d course of r-hSCF significantly increased dermal mast cell density at sites distant to those injected with the cytokine and also increased both urinary levels of the major histamine metabolite, methyl- histamine, and serum levels of mast cell alpha-tryptase. Five subjects developed areas of persistent hyperpigmentation at r-hSCF injection sites; by light microscopy, these sites exhibited markedly increased epidermal melanization and increased numbers of melanocytes. The demonstration that r-hSCF can promote both the hyperplasia and the functional activation of human mast cells and melanocytes in vivo has implications for our understanding of the role of endogenous SCF in health and disease. These findings also indicate that the interaction between SCF and its receptor represents a potential therapeutic target for regulating the numbers and functional activity of both mast cells and cutaneous melanocytes.
PMCID: PMC2192599  PMID: 8676090

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