Angiogenic Properties of NK Cells in Cancer and Other Angiogenesis-Dependent Diseases.

The pathogenesis of many serious diseases, including cancer, is closely related to disturbances in the angiogenesis process. Angiogenesis is essential for the progression of tumor growth and metastasis. The tumor microenvironment (TME) has immunosuppressive properties, which contribute to tumor expansion and angiogenesis. Similarly, the uterine microenvironment (UME) exerts a tolerogenic (immunosuppressive) and proangiogenic effect on its cells, promoting implantation and development of the embryo and placenta. In the TME and UME natural killer (NK) cells, which otherwise are capable of killing target cells autonomously, enter a state of reduced cytotoxicity or anergy. Both TME and UME are rich with factors (e.g., TGF-ß, glycodelin, hypoxia), which support a conversion of NK cells to the low/non-cytotoxic, proangiogenic CD56brightCD16low phenotype. It is plausible that the phenomenon of acquiring proangiogenic and low cytotoxic features by NK cells is not only limited to cancer but is a common feature of different angiogenesis-dependent diseases (ADDs). In this review, we will discuss the role of NK cells in angiogenesis disturbances associated with cancer and other selected ADDs. Expanding the knowledge of the mechanisms responsible for angiogenesis and its disorders contributes to a better understanding of ADDs and may have therapeutic implications.

Does Caesarean Section or Preterm Delivery Influence TGF-ß2 Concentrations in Human Colostrum?

Human colostrum (HC) is a rich source of immune mediators that play a role in immune defences of a newly born infant. The mediators include transforming growth factor ß (TGF-ß) which exists in three isoforms that regulate cellular homeostasis and inflammation, can induce or suppress immune responses, limit T helper 1 cells (Th1) reactions and stimulate secretory immunoglobulin A (IgA) production. Human milk TGF-ß also decreases apoptosis of intestinal cells and suppresses macrophage cytokine expression. The aim of the study was to determine the concentration of TGF-ß2 in HC obtained from the mothers who delivered vaginally (VD) or by caesarean section (CS), and to compare the concentrations in HC from mothers who delivered at term (TB) or preterm (PB). In this study, 56% of preterm pregnancies were delivered via CS. The concentrations of TGF-ß2 were measured in HC from 299 women who delivered in the 1st Department of Obstetrics and Gynaecology, Medical University of Warsaw: 192 (VD), 107 (CS), 251 (TB), and 48 (PB). The colostrum samples were collected within 5 days post-partum. TGF-ß2 levels in HC were measured by the enzyme-linked immunosorbent assay (ELISA) test with the Quantikine ELISA Kit-Human TGF-ß2 (cat.no. SB250). Statistical significance between groups was calculated by the Student t-test using StatSoft Statistica 13 software. The mean TGF-ß2 concentration in patients who delivered at term or preterm were comparable. The levels of TGF-ß2 in HC were higher after preterm than term being 4648 vs. 3899 ng/mL (p = 0.1244). The delivery via CS was associated with higher HC concentrations of TGF-ß2. The levels of TGF-ß2 were significantly higher in HC after CS than VD (7429 vs. 5240 ng/mL; p = 0.0017). The data from this study suggest: caesarean section was associated with increased levels of TGF-ß2 in HC. The increased levels of TGF-ß2 in HC of women who delivered prematurely require further research. Early and exclusive breast-feeding by mothers after caesarean section and premature births with colostrum containing high TGF-ß2 levels may prevent the negative impact of pathogens which often colonize the gastrointestinal tract and may reduce the risk of chronic diseases in this group of patients.

Therapeutic potential of curcumin in eye diseases.

Curcumin (diferuloylmethane) derived from the rhizome of Curcuma longa L. has been used for thousands of years in traditional Chinese medicine and Ayurvedic medicine in Asian countries to treat liver diseases, rheumatoid diseases, diabetes, atherosclerosis, infectious diseases and cancer. It exhibits a wide range of pharmacological properties, which include antioxidant, anti-inflammatory, antimutagenic, antimicrobial and anticancer activity. Herein the mechanisms of curcumin impact on oxidative stress, angiogenesis and inflammatory processes are described indicating that curcumin use may inhibit those pathological conditions and restore body homeostasis. Its effectiveness was also proved for major eye diseases. In this review, the influence of curcumin on eye diseases, such as glaucoma, cataract, age-related macular degeneration, diabetic retinopathy, corneal neovascularization, corneal wound healing, dry eye disease, conjunctivitis, pterygium, anterior uveitis are reported. The analysis of a number of clinical and preclinical investigations indicates that curcumin may be used as a therapeutic agent in the treatment of various eye disorders.

In search of chondrocyte-specific antigen.

The purpose of this work was to establish, whether rat chondrocyte associated antigen, transmembrane Tmp21 protein belonging to the p24 protein family may immunize rats and thus be included into the panel of immunogens potentially involved in cartilage pathology. For immunization of rats extract from cultured chondrocytes containing surface chondrocyte proteins suspended in incomplete Freund's adjuvant was used. Control animals were injected with incomplete Freund's adjuvant without chondrocyte extract. Morphological observations indicated that both in control and experimental animals occurred subperiosteal resorption of bone, suggesting that it arised as the response to adjuvant. In trachea, however, resorption of cartilage and inflammatory changes in the respiratory epithelium and lamina propria were present only in animals exposed to antigen. Unexpectedly, sera from immunized rats strongly reacted with other antigen, which we were able to identify by Western blot and protein sequencing as cartilage oligomeric matrix protein (COMP). COMP is attached to chondrocyte membrane by integrins and its presence in chondrocyte extract is not surprising. Antibody response to COMP raises a question whether the observed changes in tracheal cartilage and epithelium represent anti-COMP reaction or were caused by some other, no specified factors. COMP is used as the marker of osteoarthritis progression, but its role in polychondritis, cartilage pathology involving i.a. tracheal cartilage resorption remains unknown. Thus, our observations may serve as the starting point for future studies in this direction.

Antigenic and immunogenic properties of chondrocytes. Implications for chondrocyte therapeutic transplantation and pathogenesis of inflammatory and degenerative joint diseases.

In physiological conditions chondrocytes are protected from contact with immunocompetent cells by the extracellular matrix, and transplanted fragments of allogeneic cartilage are not rejected. Cartilage produced by allogeneic chondrocytes, however, evokes the immune response of the recipient and is gradually destroyed. Immunisation by allogeneic chondrocytes is induced by the contact of their surface molecules with cells of the immune system. Chondrocytes constitutively express class I and, in some species, class II major histocompatibility complex (MHC) molecules. Expression of MHC class II molecules is induced in vitro by pro-inflammatory cytokines and in vivo in the course of the rejection of transplanted allogeneic cartilage. Low level of MHC class II molecules is found on the surface of human articular chondrocytes in patients with rheumatoid arthritis and osteoarthritis. Cartilage produced by transplanted allogeneic chondrocytes is destroyed by monocytes/macrophages and cytotoxic T and natural killer (NK) cells. NK cells show spontaneous cytotoxic reactivity against isolated chondrocytes and participate in the rejection of transplanted isolated chondrocytes. Chondrocytes express molecules that can serve as potential antigens in inflammatory joint diseases. Chondrocytes express cartilage-specific membrane antigen (CH65), human cartilage glycoprotein-39 (HC gp-39), hyaluronan binding adhesion molecule CD44, thymocyte antigen-1 (Thy-1) - CD90, signal transducer - CD24, lymphocyte function-associated antigen-3 (LFA-3) - CD58, and type I transmembrane protein Tmp21. On the other hand, although chondrocytes express major histocompatibility complex (MHC) class I and class II molecules, they can also exert immunosuppressive and immunomodulatory effects on immunocompetent cells. Isolated chondrocytes do not trigger an efficient allogeneic immune response in vitro and suppress, in a contact-dependent manner, proliferation of activated T cells. This suppression is associated with the expression by chondrocytes of multiple negative regulators of immune response. Chondrocytes express programmed death-ligand (PD-L), chondromodulin-I and indoleamine 2,3-dioxygenase (IDO), molecules that promote self-tolerance and suppress the immune system.

Mouse Proepicardium Exhibits a Sprouting Response to Exogenous Proangiogenic Growth Factors in vitro.

Angiogenesis contributes to the generation of the vascular bed but also affects the progression of many diseases, such as tumor growth. Many details of the molecular pathways controlling angiogenesis are still undefined due to the lack of appropriate models. We propose the proepicardial explant as a suitable model for studying certain aspects of angiogenesis. The proepicardium (PE) is a transient embryonic structure that contains a population of undifferentiated endothelial cells (ECs) forming a vascular net continuous with the sinus venosus. In this paper, we show that PE explants give rise to CD31-positive vascular sprouts in the presence of basic fibroblast growth factor (bFGF) and 2 isoforms of vascular endothelial growth factor A (VEGF-A), i.e. VEGF-A120 and VEGF-A164. Vascular sprouts exhibit differences in number, length, thickness and the number of branches, depending on the combination of growth factors used. Moreover, the ECs of the sprouts express various levels of mRNA for Notch1 and its ligand Dll4. Additionally, stimulation with bFGF/VEGF-A164 upregulates the expression of Lyve-1 antigen in the ECs in the sprouts. In summary, we present a new model for angiogenesis studies involving mouse PE as a source of ECs. We believe that our model may act as a supplementary assay for angiogenesis studies along with the existing models.

Angiomodulatory properties of some antibiotics and Tolpa Peat Preparation.

UNLABELLED: Deterioration of the immune system due to antibiotic therapy can be restored by immunomodulator application. In this paper we estimate the effect of ampicillin, amikacin, doxycycline, rifampicin, rifamycine and immunomodulator Tolpa Peat Preparation (TPP) on neovascular reaction induced in murine skin by human mononuclear cells (MNC) injection. MNC originating from 15 healthy volunteers were injected intradermally to Balb/c mice. Antibiotics (3, 15, or 75 mg/kg of body weight) alone or with TPP (10 mg/kg of body weight) were administrated subcutaneously to mice on three consecutive days. The number of newly formed blood vessels was measured in dissection microscope 72 hours after cell injection. RESULTS: TPP stimulated angiogenic activity of MNC at the dose 5 and 10 mg/kg. Rifamycine exerted strong stimulatory action, ampicillin slightly stimulated immune response, while doxycycline and rifampicin downregulated it. Amikacin did not influence the results of angiogenesis tests. Studied antibiotics (15 mg/kg), except rifamycine, inhibit the angiostimulatory effect of the tested immunomodulator. TPP should be applied after antibiotic therapy to maintain its stimulatory effect and restore proper host immune function.

A natural herbal remedy modulates angiogenic activity of bronchoalveolar lavage cells from sarcoidosis patients.

Sarcoidosis is a systemic inflammatory disease with abnormally high angiogenic activity of inflammatory cells. Reumaherb preparation consisting of three herbs: Echinacea purpurea, Harpagophytum procumbens, and Filipendula ulmaria, and it exerts anti-inflammatory, antioxidant, and analgesic activity and stimulates regenerative and immunological processes. The aim of this paper was to estimate the effect of Reumaherb on immunological angiogenesis induced by bronchoalveolar lavage (BAL) cells collected from six patients with sarcoidosis and grafted into Balb/c mice skin. After grafting, the animals were fed for three days with 0.6 or 1.2 mg of Reumaherb (calculated from recommended human daily dose) daily, suspended in 40 µl of water, or 40 µl of water alone (control group). A significant reduction of newly formed blood vessels was obtained in four cases for 1.2 mg and in three cases for 0.6 mg daily dose of this remedy. Thus, we hypothesise that Reumaherb promotes anti-angiogenic activity and may potentially be used in diseases associated with excessive blood vessel formation.

Reactive oxygen species and synthetic antioxidants as angiogenesis modulators: Clinical implications.

Angiogenesis is important for normal functioning of organism and its disturbances are observed in many diseases, called angiogenesis-related states. Reactive oxygen species (ROSs) play an important role in physiology, but high level of cellular ROSs is cytotoxic and mutagenic for the cells, i.e. it can lead to oxidative stress. In this review we discuss close relationship between ROSs and angiogenesis process. Substances counteracting free radicals or their action and oxidative stress are known as antioxidants. We postulate that antioxidants, by affecting angiogenesis, may modulate therapy results in the case of angiogenesis-related disease. Herein, we present some antioxidant preparations of synthetic (N-acetylcysteine, curcumin and its analogs, Probucol, oleane tripertenoid, EGCG synthetic analogs) and nature-identical (vitamin E and C) origin. Then, we analyze their angiogenic properties and their multidirectional molecular effect on angiogenesis. Most preparations reduce neovascularization and diminish the level of proangiogenic molecules, downregulating signaling pathways related to angiogenesis. Moreover, we discuss studies concerning anticancer properties of presented synthetic antioxidants and their application in several angiogenesis-related diseases. We conclude that therapy in angiogenesis-related diseases should be planned with consideration of the angiogenic status of the patient.

Angiomodulatory properties of Rhodiola spp. and other natural antioxidants.

Disturbances of angiogenesis and oxidative stress can lead to many serious diseases such as cancer, diabetes or ischemic heart disease. Substances neutralizing oxidative stress are known as antioxidants. They can affect angiogenesis process also, and thus, they modulate therapy results. Antioxidants become more and more frequently used in order to maintain homeostasis of the organism and diminish the risk of disease. Herein, we introduce some antioxidant preparations of natural plant origin (Rhodiola, Aloe vera, Resveratrol, Echinacea, Plumbagin) and antioxidant supplements (Padma 28, Reumaherb, Resvega). Analyses of their angiogenic properties, their multidirectional molecular effect on angiogenesis as well as medical application are within the scope of this review. Most of presented preparations down regulate neovascularization. They can be safely administered to patients with abnormally high angiogenesis. Rhodiola modulates, and Echinacea, Aloe vera and Plumbagin inhibit tumour-related angiogenesis in vitro and in vivo (animal models). Resveratrol and Resvega reduce neovascularization in the eye and may be applicable in eye disorders. Padma 28 preparation exhibits angioregulatory activity, decreasing high angiogenesis of cancer cells and increasing physiological angiogenesis, therefore can be used in therapy of patients with various disturbances of angiogenesis. Antioxidant application in the case of angiogenesis-related diseases should take into consideration angiogenic status of the patient.

3-D reconstruction and multiple marker analysis of mouse proepicardial endothelial cell population.

BACKGROUND: The proepicardium (PE), a transient embryonic structure crucial for the development of the epicardium and heart, contains its own population of endothelial cells (ECs). The aim of our study was to determine the pattern, anatomical orientation and phenotypic marker expression of the endothelial cell network within the PE. RESULTS: Immunohistochemical findings revealed that proepicardial ECs express both early and late EC-specific markers such as CD31, Flk-1, Lyve-1 and Tie-2 but not SCL/Tal1, vWF, Dll4 or Notch1. Proepicardial ECs are present in the vicinity of the sinus venosus (SV) and form a continuous network of vascular sprouts/tubules connected with the SV endothelium, with Ter-119-positive erythroblasts in the vascular lumina. CONCLUSIONS: On the basis of our results, we postulate the existence of a continuous network of ECs in the PE, exhibiting connection and/or patency with the SV and forming vessels/tubules/strands. Marker expression suggests that ECs are immature and undifferentiated, which was also confirmed with a transmission electron microscopy (TEM) analysis. Our results deliver new data for a better understanding of the nature of proepicardial ECs.

Vasculogenic and hematopoietic cellular progenitors are scattered within the prenatal mouse heart.

Vasculogenesis and hematopoiesis are co-localized in the embryonic body, but precise phenotypes of the cells contributing to these processes are not defined. The aim of this study was to characterize phenotypic profiles and location of putative vasculogenic and hematopoietic cellular progenitors in the embryonic mouse heart. Confocal microscopy, as well as ultrastructural and stereomicroscopic analyses, was performed on immunohistochemical whole-mount-stained or sectioned hearts at stages 11.5-14 dpc. A FASC analysis was conducted to quantify putative vasculogenic and hematopoietic cells. We found subepicardial blood islands in the form of foci of accumulation of cells belonging to erythroblastic and megakaryocytic lineages at various stages of maturation, exhibiting phenotypes: GATA2(+)/CD41(+), GATA2(-)/CD41(+), GATA2(+)/CD71(-), GATA2(-)/CD71(+), Fli1(+)/CD71(+), Fli1(-)/CD71(+), with a majority of cells expressing the Ter119 antigen, but none of them expressing Flk1. The subepicardium and the outflow tract endothelium were recognized to be the areas where progenitor cells were scattered or adjoining the endothelial cells. These progenitor cells were characterized as possessing the following antigens: CD45(+)/Fli1(+), CD41(+)/Flk1(+), Flk1(+)/Fli1(+). A FACS analysis demonstrated that the CD41/Flk1 double-positive population of cells constituted 2.68% of total cell population isolated from 12.5 dpc hearts. Vessels and tubules were positive for CD31, Flk1, Fli1, Tie2, including blood islands endothelia. The endocardial wall endothelia were found to function as an anchoring apparatus for megakaryocytes releasing platelets into the cardiac cavities. Phenotypic characteristics of vasculogenic (Flk1(+)/Fli1(+)) and hematopoietic (GATA2(+)/CD71(+), CD41(+)/GATA2(+)) progenitors, as well as the putative hemogenic endothelium (Flk1(+)/CD41(+)) in embryonic mouse hearts, have been presented. Cardiac blood islands, the subepicardium and endothelium of the outflow tract cushions have been defined as areas where these progenitor cells can be found.

The effect of anti-inflammatory and antimicrobial herbal remedy PADMA 28 on immunological angiogenesis and granulocytes activity in mice.

UNLABELLED: PADMA 28 is a herbal multicompound remedy that originates from traditional Tibetan medicine and possesses anti-inflammatory, antioxidant, antimicrobial, angioprotecting, and wound healing properties. The aim of the present study was to evaluate the influence of this remedy on immunological angiogenesis and granulocytes metabolic activity in Balb/c mice. Mice were fed daily, for seven days, with 5.8 mg of PADMA (calculated from recommended human daily dose) or 0.085 mg (dose in the range of active doses of other herbal extracts studied by us previously). RESULTS: Highly significant increase of newly formed blood vessels number in ex vivo cutaneous lymphocyte-induced angiogenesis test (LIA) after grafting of Balb/c splenocytes from both dosage groups to F1 hybrids (Balb/c × C3H); increase of blood lymphocytes and granulocytes number only in mice fed with lower dose of remedy; and significant suppression of metabolic activity (chemiluminescence test) of blood granulocytes in mice fed with higher dose of PADMA. CONCLUSION: PADMA 28 behaves as a good stimulator of physiological angiogenesis, but for this purpose it should be used in substantially lower doses than recommended by producers for avoiding the deterioration of granulocyte function.

N-acetylcysteine inhibits IL-8 and MMP-9 release and ICAM-1 expression by bronchoalveolar cells from interstitial lung disease patients.

N-acetylcysteine (NAC), owing to its antioxidant, mucolytic and anti-inflammatory properties, is used in the treatment of various pulmonary disorders. However, the direct effects of NAC on bronchoalveolar lavage (BAL) cells from patients suffering from interstitial lung diseases have not yet been studied. Therefore, the aim of the present work was to evaluate the effect of NAC on interleukin-8 (IL-8) and matrix metalloproteinase-9 (MMP-9) production as well as intercellular cell adhesion molecule-1 (ICAM-1) expression by BAL cells from interstitial lung diseases. The study was performed on BAL cells from nine patients with interstitial lung disease: four patients with idiopathic pulmonary fibrosis (IPF) and five patients with sarcoidosis. Cultured unstimulated BAL cells were treated with increasing doses of NAC (1-30 mM). Production of IL-8 and MMP-9 was evaluated by specific enzyme-linked immuno-sorbent assays and ICAM-1 expression was studied by immunohistochemistry. NAC exerted a dose-dependent inhibitory effect on IL-8 and MMP-9 release and ICAM- expression by BAL macrophages and lymphocytes from patients with IPF and sarcoidosis. In conclusion, NAC inhibits production of factors playing a key role in the etiopathogenesis of interstitial lung diseases, thus suggesting its possible therapeutic potency in the treatment of these disorders.

Chondrocyte-specific phenotype confers susceptibility of rat chondrocytes to lysis by NK cells.

Normal chondrocytes are targets for natural killer (NK) cells. Since the mechanism of this phenomenon remains unknown, the present study was aimed at testing whether it is associated with chondrocyte-specific phenotype defined as ability of cartilage cells to produce sulfated glycosaminoglycans (GAG) and express collagen II and aggrecan mRNA. Lysis of rat epiphyseal chondrocytes by syngeneic spleen mononuclear cells (SMCs) was evaluated by (51)Cr-release assay. Loss of chondrocyte phenotype following long-term culture resulted in their decreased susceptibility to lysis. Similar effect was also observed after suppression of chondrocyte phenotype by TNF. On the other hand, stimulation of cartilage-specific matrix component synthesis by IGF-1 resulted in increased chondrocyte killing and exogenous chondroitin sulfate A stimulated NK cell-mediated cytotoxicity against chondrocytes and human K562 cells. This suggests that chondrocyte susceptibility to lysis by NK cells depends on chondrocyte-specific phenotype, especially sulfated GAG production.