Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF

Src-family kinases mediate an outside-in signal necessary for β2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin–IgG fusion protein, by a mechanism that involved β2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(β2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an ‘outside-in’ signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented β2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin–IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck−/−fgr−/− and hck−/−fgr−/−lyn−/− mice showed significant impairment of β2-integrin-mediated adhesion to CHO-E. Moreover, the expression of β2 integrin activation epitopes at the sites of cell–cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a β2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for β2 integrin function in PMN tethered by E-selectin

Fucans, but Not Fucomannoglucuronans, Determine the Biological Activities of Sulfated Polysaccharides from Laminaria saccharina Brown Seaweed

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed

SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells.

In human PMN (polymorphonuclear cells), challenged by P-selectin, the beta2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108-116]. This suggested that an SRC-dependent outside-in signalling strengthens the beta2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock beta2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor beta2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common beta2 chain, by a combination of antibodies against alphaL and alphaM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by beta2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by beta2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize beta2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process

Src family kinases mediate neutrophil adhesion to adherent platelets

Polymorphonuclear leukocyte (PMN)–platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of “outside-in” signaling by Src-family tyrosine kinases (SFKs) in the regulation of αMβ2-integrin–dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in β2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from αMβ2(−/−), hck(−/−)fgr(−/−), and hck(−/−)fgr(−/−)lyn(−/−) mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of αMβ2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for αMβ2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin–PSGL-1–mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease

A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds

The anti-inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans obtained from nine species of brown algae were studied in order to examine the influence of fucoidan origin and composition on their biological activities. All fucoidans inhibited leucocyte recruitment in an inflammation model in rats, and neither the content of fucose and sulfate nor other structural features of their polysaccharide backbones significantly affected the efficacy of fucoidans in this model. In vitro evaluation of P-selectin-mediated neutrophil adhesion to platelets under flow conditions revealed that only polysaccharides from Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, and Ascophyllum nodosum could serve as P-selectin inhibitors. All fucoidans, except that from Cladosiphon okamuranus carrying substantial levels of 2-O-alpha-D-glucuronopyranosyl branches in the linear (1-->3)-linked poly-alpha-fucopyranoside chain, exhibited anticoagulant activity as measured by activated partial thromboplastin time whereas only fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. evanescens displayed strong antithrombin activity in a platelet aggregation test. The last fucoidans potently inhibited human umbilical vein endothelial cell (HUVEC) tubulogenesis in vitro and this property correlated with decreased levels of plasminogen-activator inhibitor-1 in HUVEC supernatants, suggesting a possible mechanism of fucoidan-induced inhibition of tubulogenesis. Finally, fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. vesiculosus strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets, an effect which might have critical implications in tumor metastasis. The data presented herein provide a new rationale for the development of potential drugs for thrombosis, inflammation, and tumor progression.Fil: Cumashi, Albana. Universita degli Studi G. D Annunzio; ItaliaFil: Ushakova, Natalia A.. Academia Rusa de Ciencias Médicas; RusiaFil: Preobrazhenskaya, Marina E.. Academia Rusa de Ciencias Médicas; RusiaFil: D'Incecco, Armida. Universita degli Studi G. D Annunzio; ItaliaFil: Piccoli, Antonio. Consorzio Mario Negri Sud; ItaliaFil: Totani, Licia. Consorzio Mario Negri Sud; ItaliaFil: Tinari, Nicola. Universita degli Studi G. D Annunzio; ItaliaFil: Morozevich, Galina E.. Academia Rusa de Ciencias Médicas; RusiaFil: Berman, Albert E.. Academia Rusa de Ciencias Médicas; RusiaFil: Bilan, María. Academia Rusa de Ciencias; RusiaFil: Usov, Anatolii I.. Academia Rusa de Ciencias; RusiaFil: Ustyuzhanina, Nadezhda E.. Academia Rusa de Ciencias; RusiaFil: Grachev, Alexey A.. Academia Rusa de Ciencias; RusiaFil: Sanderson, Craig J.. Scottish Association for Marine Sciences; Reino UnidoFil: Kelly, Maeve. Scottish Association for Marine Sciences; Reino UnidoFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Iacobelli, Stefano. Universita degli Studi G. D Annunzio; ItaliaFil: Nifantiev, Nikolay E.. Academia Rusa de Ciencias; Rusi

Effect of polysaccharide preparations on tumor growth and angiogenesis <i>in vivo</i>.

<p>C57BL/6 (B6) mice were injected with 500 µl of Matrigel containing 1×10⁵ B16-F10 cells in PBS or 100 µg of a non-fractionated polysaccharide mixture <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b>. After 6–7 days, tumors were removed and hemoglobin content was evaluated by using the Drabkin colorimetric method. Results are expressed as the amount of hemoglobin (mg)/Matrigel weight (mg) (<b>A</b>) (**<i>P</i><0.01). (<b>B</b>) Flow cytometry analysis of the frequency of CD34⁺ endothelial cells on Matrigel plugs embedded with B16 melanoma cells. (**<i>P</i><0.01) (<b>C</b>) <i>In vitro</i> cell growth of B16 melanoma cells exposed to 100 µg/ml of <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b>. Data are the mean ± SEM of three independent experiments. (<b>D</b>) B6 mice were injected with 500 µl Matrigel containing 1×10⁵ B16-F10 cells. <b>L.s.-P</b> or its fractions <b>L.s.-1.0</b> and <b>L.s.-1.25</b> were injected <i>i.p.</i> at doses of 50 mg/kg every 3 days and compared to control (PBS). Tumors were removed on day 21 post-implantation, photographed (<b>D</b>) and analyzed for CD31⁺ associated blood vessels (<b>E</b>), microvessel density (<b>F</b>) and weight (<b>G</b>). (*<i>P</i><0.05; **<i>P</i><0.01).</p