Endothelial Cell Interactions Research Articles

Quantitative Imaging-Based Examination of Pericytes Controlling Endothelial Growth Dynamics and Angiogenesis.

Microvascular endothelial cell-mural cell interactions are instrumental in modulating both physiological and pathologic angiogenesis. Pericyte-endothelial cell communication through direct physical associations and secreted effectors comprises a bidirectional signal array that regulates vascular maturation and integrity. As endothelial cell proliferation, migration, and morphogenesis are key elements of vascular growth and remodeling during angiogenesis, we have developed novel preclinical systems for studying the roles of endothelial-mural cell dynamics on cell cycle entry and angiogenic activity in vitro. These coculture models not only enable evaluation of endothelial cell-pericyte "cross talk" but also allow for the quantitative analysis of both heterotypic contact-dependent and contact-independent cell cycle progression in either cell population, as well as angiogenic sprouting in three-dimensional vascular networks. Cells actively proliferating in two-dimensional assays can be labeled via incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into their DNA. Additionally, each cell population can be vitally labeled with a variety of cell-specific and/or membrane-permeant lipophilic dyes prior to coculture, such as DiO, or through immunofluorescence of mural or endothelial cell-specific markers after cellular fixation and/or permeabilization. Ultimately, this experimental approach can be used to investigate cellular contact-dependent and soluble mechanisms mediating mural-endothelial cell interactions, which may be instrumental in microvascular development and remodeling in vivo.

Putative CD133 + melanoma cancer stem cells induce initial angiogenesis in vivo

Tumor angiogenesis is essential for tumor growth and metastasis, and is regulated by a complex network of various types of cells, chemokines, and stimulating factors. In contrast to sprouting angiogenesis, tumor angiogenesis is also influenced by hypoxia, inflammation, and the attraction of bone-marrow-derived cells. Recently, cancer stem cells have been reported to mimic vascularization by differentiating into endothelial cells and inducing vessel formation. In this study, the influence of cancer stem cells on initial angiogenesis was evaluated for the metastatic melanoma cell line D10. Following flow cytometry, CD133+ and CD133− cells were isolated using magnetic cell separation and different cell fractions were transferred to porcine gelatin sponges, which were implanted into the dorsal skinfold chamber of immunocompromised mice. Angiogenesis was analyzed based on microvessel density over a 10-day period using in vivo fluorescence microscopy, and the results were verified using immunohistology. CD133+ D10 cells showed a significant induction of early angiogenesis in vivo, contrary to CD133− D10 cells, unsorted D10 cells, and negative control. Neovascularization was confirmed by visualizing endothelial cells by immunohistology using an anti-CD31 antibody. Because CD133+ cells are rare in clinical specimens and hardly amenable to functional assays, the D10 cell line provides a suitable model to study the angiogenic potential of putative cancer stem cells and the leukocyte–endothelial cell interaction in the dorsal skinfold chamber in vivo. This cancer stem cell model might be useful in the development and evaluation of therapeutic agents targeting tumors.

Human severe sepsis cytokine mixture increases β2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro.

IntroductionSepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro.MethodsThe concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm2).ResultsEight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either β2 (CD18), αL/β2 (CD11α/CD18; LFA-1) or αM/β2 (CD11β/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion.ConclusionsHuman SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a β2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.

Mesenchymal stromal cells support endothelial cell interactions in an intramuscular islet transplantation model.

BackgroundMesenchymal stromal cells (MSC) have been under investigation for a number of therapies and have lately been in focus as immunosuppressive actors in the field of transplantation. Herein we have extended our previously published in vitro model of MSC-islets in an experimental setting of islet transplantation to the abdominal muscle.Human islets coated with luciferase-GFP transduced human MSC were transplanted to the abdomen muscle tissue of NOD-scid ILR2γnull mice and cellular interactions were investigated by confocal microscopy.ResultsThe MSC reduced fibrotic encapsulation and facilitated endothelial cell interactions. In particular, we show a decreased fraction of αSMA expressing fibrotic tissue surrounding the graft in presence of MSC-islets compared to islets solely distributed into the muscle tissue. Also, in the presence of MSC, human islet endothelial cells migrated from the center of the graft out into the surrounding tissue forming chimeric blood vessels with recipient endothelial cells. Further, in the graft periphery, MSC were seen interacting with infiltrating macrophages.ConclusionsHere, in our experimental in vivo model of composite human islets and luciferase-GFP-transduced human MSC, we enable the visualization of close interactions between the MSC and the surrounding tissue. In this model of transplantation the MSC contribute to reduced fibrosis and increased islet endothelial cell migration. Furthermore, the MSC interact with the recipient vasculature and infiltrating macrophages.Electronic supplementary materialThe online version of this article (doi:10.1186/s40340-015-0010-9) contains supplementary material, which is available to authorized users.

β-elemene inhibits monocyte–endothelial cells interactions via reactive oxygen species/MAPK/NF-κB signaling pathway in vitro

The recruitment of monocytes to the active endothelial cells is an early step in the formation of atherosclerotic lesions; therefore, the inhibition of monocyte–endothelial cells interactions may serve as a potential therapeutic strategy for atherosclerosis. Recent studies suggest that β-elemene can protect against atherosclerosis in vivo and vitro; however, the mechanism underlying the anti-atherosclerotic effect by β-elemene is not clear yet. In this study, we aimed to investigate the effects of β-elemene on the monocyte–endothelial cells interactions in the initiation of atherosclerosis in vitro. Our results showed that β-elemene protects human umbilical vein endothelial cells (HUVECs) from hydrogen peroxide-induced endothelial cells injury in vitro. Besides, this molecule inhibits monocyte adhesion and transendothelial migration across inflamed endothelium through the suppression of the nuclear factor-kappa B-dependent expression of cell adhesion molecules. Further, β-elemene decreases generation of reactive oxygen species (ROS) and prevents the activation of mitogen-activated protein kinase (MAPK) signaling pathway in HUVECs. In conclusion, this study would provide a new pharmacological evidence of the significance of β-elemene as a future drug for prevention and treatment of atherosclerosis.

Differential effects of anti-TNF-α and anti-IL-12/23 agents on human leukocyte–endothelial cell interactions

Enhanced leukocyte recruitment is an inflammatory process that occurs during early phases of the vascular dysfunction that characterises atherosclerosis. We evaluated the impact of anti-TNF-α (adalimumab, infliximab and etanercept) and anti-IL-12/23 (ustekinumab) on interactions between human leukocytes and endothelial cells in a flow chamber that reproduced in vivo conditions. Clinical concentrations of anti-TNF-α were evaluated on the leukocyte recruitment induced by a variety of endothelial (TNF-α, interleukin-1β, lymphotoxin-α and angiotensin-II) and leukocyte (PAF, IL-12 and IL-23) stimuli related to inflammation and atherosclerosis. Treatment with anti-TNF-α, even before or after establishing the inflammatory situation induced by TNF-α, diminished leukocyte–endothelial cell interactions induced by this stimuli. Our results also implicated adhesion molecules (ICAM-1, VCAM-1 and E-selectin) in the actions of anti-TNF-α in terms of leukocyte adhesion to endothelium. However, anti-TNF-α drugs did not influence the actions of interleukin-1β, but prevented those of lymphotoxin-α and angiotensin-II. However, once established, inflammatory response elicited by the latter three stimuli could not be reversed. Pre-treatment with anti-TNF-α, also prevented leukocyte actions induced by IL-23 on PBMC rolling flux and rolling velocity and by IL-12 on PMN adhesion. Ustekinumab exhibited a more discreet profile, having no effect on leukocyte recruitment induced by any of the endothelial stimuli, while blocking the effects of IL-23 on leukocyte activation and those of IL-12 on PMN adhesion and PAF on PBMC rolling velocity. These findings endorse the idea that biological anti-inflammatory drugs, in particular anti-TNF-α, have the capacity to influence cardiovascular risk accompanying psoriasis and rheumatoid arthritis by ameliorating vascular inflammation.

Role of protein kinase CK2 in the dynamic interaction of platelets, leukocytes and endothelial cells during thrombus formation

IntroductionThrombus formation is a complex process, which is characterized by the dynamic interaction of platelets, leukocytes and endothelial cells. The activation of these cells is strictly mediated by different phospho-regulated signaling pathways. Recently, it has been reported that inhibition of protein kinase CK2 affects platelet function by suppressing phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K) signaling. Based on this finding, we herein analyzed whether CK2 acts as a crucial regulator of thrombus formation. Materials and methodsWe examined the effect of CK2 inhibition on platelet activation and aggregation, the formation of platelet-leukocyte aggregates (PLA), the endothelial expression of von Willebrand factor (vWF), intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, and the subcellular localization of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and phospho-p65 in human dermal microvascular endothelial cells (HDMEC). Dorsal skinfold chambers were prepared in BALB/c mice to analyze in vivo the effect of CK2 inhibition on photochemically induced thrombus formation using intravital fluorescence microscopy. ResultsCK2 inhibition by CX-4945 suppressed adenosin diphosphate (ADP)- and proteinase-activated receptor-1-peptide (PAR-1-AP)-stimulated platelet aggregation, which was associated with down-regulation of P-selectin, GPIIb/IIIa and a reduced formation of PLA. Expression and secretion of vWF was diminished in CX-4945-treated HDMEC. Moreover, CK2 inhibition attenuated the endothelial expression of VCAM-1, whereas the expression of ICAM-1 was not affected. Finally, CX-4945-treated mice exhibited a significantly delayed photochemically induced thrombus formation when compared to vehicle-treated controls. ConclusionThese results indicate that CK2 is a pleiotropic regulator of thrombus formation, affecting multiple interactions of platelets, leukocytes and endothelial cells.

Organotypic brain slice cultures as a model to study angiogenesis of brain vessels.

Brain vessels are the most important structures in the brain to deliver energy and substrates to neurons. Brain vessels are composed of a complex interaction between endothelial cells, pericytes, and astrocytes, controlling the entry of substrates into the brain. Damage of brain vessels and vascular impairment are general pathologies observed in different neurodegenerative disorders including e.g., Alzheimer's disease. In order to study remodeling of brain vessels, simple 3-dimensional in vitro systems need to be developed. Organotypic brain slices of mice provide a potent tool to explore angiogenic effects of brain vessels in a complex 3-dimensional structure. Here we show that organotypic brain slices can be cultured from 110 μm thick sections of postnatal and adult mice brains. The vessels are immunohistochemically stained for laminin and collagen IV. Co-stainings are an appropriate method to visualize interaction of brain endothelial cells with pericytes and astrocytes in these vessels. Different exogenous stimuli such as fibroblast growth factor-2 or vascular endothelial growth factor induce angiogenesis or re-growth, respectively. Hyperthermia or acidosis reduces the vessel density in organotypic slices. In conclusion, organotypic brain slices exhibit a strong vascular network which can be used to study remodeling and angiogenesis of brain vessels in a 3-dimensional in vitro system.

Endothelial LSP1 Modulates Extravascular Neutrophil Chemotaxis by Regulating Nonhematopoietic Vascular PECAM-1 Expression.

During inflammation, leukocyte-endothelial cell interactions generate molecular signals that regulate cell functions. The Ca(2+)- and F-actin-binding leukocyte-specific protein 1 (LSP1) expressed in leukocytes and nonhematopoietic endothelial cells is pivotal in regulating microvascular permeability and leukocyte recruitment. However, cell-specific function of LSP1 during leukocyte recruitment remains elusive. Using intravital microscopy of cremasteric microvasculature of chimeric LSP1-deficient mice, we show that not neutrophil but endothelial LSP1 regulates neutrophil transendothelial migration and extravascular directionality without affecting the speed of neutrophil migration in tissue in response to CXCL2 chemokine gradient. The expression of PECAM-1-sensitive α6β1 integrins on the surface of transmigrated neutrophils was blunted in mice deficient in endothelial LSP1. Functional blocking studies in vivo and in vitro elucidated that α6β1 integrins orchestrated extravascular directionality but not the speed of neutrophil migration. In LSP1-deficient mice, PECAM-1 expression was reduced in endothelial cells, but not in neutrophils. Similarly, LSP1-targeted small interfering RNA silencing in murine endothelial cells mitigated mRNA and protein expression of PECAM-1, but not ICAM-1 or VCAM-1. Overexpression of LSP1 in endothelial cells upregulated PECAM-1 expression. Furthermore, the expression of transcription factor GATA-2 that regulates endothelial PECAM-1 expression was blunted in LSP1-deficient or LSP1-silenced endothelial cells. The present study unravels endothelial LSP1 as a novel cell-specific regulator of integrin α6β1-dependent neutrophil extravascular chemotactic function in vivo, effective through GATA-2-dependent transcriptional regulation of endothelial PECAM-1 expression.

CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte–endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation.

Roles of YAP in mediating endothelial cell junctional stability and vascular remodeling.

Angiogenesis is a complex process involving dynamic interaction of various cell to cell interactions. Endothelial cell interactions regulated by growth factors, inflammatory cytokines, or hemodynamic stress are critical for balancing vascular quiescence and activation. Yes-associated protein (YAP), an effector of Hippo signaling, is known to play significant roles in maintaining cellular homeostasis. However, its role in endothelial cells for angiogenic regulation remains relatively unexplored. We demonstrated the critical role of YAP in vascular endothelial cells and elucidated the underlying molecular mechanisms involved in angiogenic regulation of YAP. YAP was expressed in active angiogenic regions where endothelial cell junctions were relatively loosened. Consistently, YAP subcellular localization and activity were regulated by VE-cadherin-mediated PI3K/Akt pathway. YAP thereby regulated endothelial sprouting via angiopoietin-2 expression. These results provide an insight into a model of coordinating endothelial junctional stability and angiogenic activation through YAP. [BMB Reports 2015; 48(8): 429-430]

Abstract 4158: Novel PAK4-mediated regulation of endothelial CXCL1/CXCR2 signaling and angiogenesis in glioblastoma

Abstract Increased endothelial cell proliferation and aberrant neovascularization are pathological hallmarks of glioblastoma that correlate with clinical outcome. Recent studies have also implicated ionizing radiation (IR)-induced angiogenesis as a potential cause for tumor recurrence and metastases in several malignancies. Its impact in gliomas has not been characterized. Factors that drive endothelial proliferation, angiogenesis and interaction of endothelial cells with tumor cells in the context of radiotherapy are potentially novel targets for glioma therapy. Our previous studies demonstrated high levels of expression of p21-activated kinase 4 (PAK4) in gliomas; analyses of tumor cell medium (CM) prepared from shRNA-mediated PAK4-knockdown cells using an angiogenesis antibody array showed a significant decrease in key angiogenic factors including CXCL1. Treatment of human brain microvascular endothelial cells (HBMECs) with CM from PAK4-knockdown cells (PAK4-kd CM) significantly inhibited proliferation, migration and microtubule formation compared with CM from control and scrambled vector-expressing glioma cells. PAK4-kd CM treatment also suppressed CXCR2 activation and decreased the expression levels of p-STAT3, VEGF, MMP-2 and cyclinD1 in HBMECs. Additionally, treatment with CM from ionizing radiation (IR, 8 Gy)-treated cells resulted in elevated CXCL1, MMP-2 and VEGF levels in HBMECs, and induced high levels of endothelial capillary network formation on matrigel, indicating an IR-induced angiogenesis in these cells. Conversely, PAK4-kd inhibited IR-induced angiogenesis and decreased p-STAT3, VEGF and MMP-2 levels in IR-treated HBMECS. Further, in vivo experiments with orthotopic intracranial tumors in nude mice showed growth suppression in PAK4-knockdown tumors compared to control tumors. Immunohistochemical analyses of brain tumor sections revealed a significant decrease in CXCL1, VEGF and MMP-2 in PAK4.sh tumors. In summary, our studies emphasize a novel regulation of angiogenic switch and endothelial CXCL1/CXCR2 signaling by PAK4 in tumor cells, thereby, suggests the role of PAK4 as a critical mediator of tumor-endothelial cell interactions in glioblastoma. Abrogation of key angiogenic signaling after IR treatment and inhibition of in vivo tumor growth by PAK4 suppression indicates its therapeutic potential in the treatment of glioma. Citation Format: Divya Kesanakurti, Jihong Xu, Alessandro Canella, Prabhakaran Nagarajan, Balveen Kaur, Vinay K. Puduvalli. Novel PAK4-mediated regulation of endothelial CXCL1/CXCR2 signaling and angiogenesis in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4158. doi:10.1158/1538-7445.AM2015-4158

Dysregulation of the endothelium following Staphylococcus aureus infection.

The cardiovascular system is typically a sterile environment; however entry of a microorganism into the circulation can cause potentially life threatening cardiac and/or vascular disease. Staphylococcus aureus endothelial cell interactions are arguably the most important interactions in the pathogenesis of cardiovascular infection. These interactions can trigger cardiac valve destruction in the case of endocarditis, multi-organ dysfunction in the case of sepsis and coagulopathy. Here, we review the interactions between S. aureus and endothelial cells and discuss the implications of these interactions in the progression of cardiovascular infection.

αvβ3 Integrins Mediate Flow-Induced NF-κB Activation, Proinflammatory Gene Expression, and Early Atherogenic Inflammation

Endothelial cell interactions with transitional matrix proteins, such as fibronectin, occur early during atherogenesis and regulate shear stress-induced endothelial cell activation. Multiple endothelial cell integrins bind transitional matrix proteins, including α5β1, αvβ3, and αvβ5. However, the role these integrins play in mediating shear stress-induced endothelial cell activation remains unclear. Therefore, we sought to elucidate which integrin heterodimers mediate shear stress-induced endothelial cell activation and early atherogenesis. We now show that inhibiting αvβ3 integrins (S247, siRNA), but not α5β1 or αvβ5, blunts shear stress-induced proinflammatory signaling (NF-κB, p21-activated kinase) and gene expression (ICAM1, VCAM1). Importantly, inhibiting αvβ3 did not affect cytokine-induced proinflammatory responses or inhibit all shear stress-induced signaling, because Akt, endothelial nitric oxide synthase, and extracellular regulated kinase activation remained intact. Furthermore, inhibiting αv integrins (S247), but not α5 (ATN-161), in atherosclerosis-prone apolipoprotein E knockout mice significantly reduced vascular remodeling after acute induction of disturbed flow. S247 treatment similarly reduced early diet-induced atherosclerotic plaque formation associated with both diminished inflammation (expression of vascular cell adhesion molecule 1, plaque macrophage content) and reduced smooth muscle incorporation. Inducible, endothelial cell-specific αv integrin deletion similarly blunted inflammation in models of disturbed flow and diet-induced atherogenesis but did not affect smooth muscle incorporation. Our studies identify αvβ3 as the primary integrin heterodimer mediating shear stress-induced proinflammatory responses and as a key contributor to early atherogenic inflammation.

Inhibition of protein kinase CK2 suppresses tumor necrosis factor (TNF)-α-induced leukocyte–endothelial cell interaction

Inflammatory endothelial processes are regulated by the nuclear factor-κB (NF-κB) pathway, which involves phosphorylation of p65. Because p65 is a substrate of CK2, we herein investigated, whether this pleiotropic protein kinase may be a beneficial anti-inflammatory target. For this purpose, we analyzed in human dermal microvascular endothelial cells (HDMEC) the effect of CK2 inhibition by quinalizarin and CX-4945 on cell viability, adhesion molecule expression and NF-κB pathway activation. Leukocyte binding to HDMEC was assessed in an in vitro adhesion assay. Dorsal skinfold chambers in BALB/c mice were used to study leukocyte–endothelial cell interaction and leukocyte transmigration by means of repetitive intravital fluorescence microscopy and immunohistochemistry. We found that quinalizarin and CX-4945 effectively suppressed the activity of CK2 in HDMEC without affecting their viability. This was associated with a significant down-regulation of tumor necrosis factor (TNF)-α-induced E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression due to a reduction of shuttling, phosphorylation and transcriptional activity of the NF-κB complex. In consequence, leukocyte binding to quinalizarin- and CX-4945-treated HDMEC was diminished. Finally, CX-4945 treatment significantly decreased the numbers of adherent and transmigrated leukocytes in dorsal skinfold chambers exposed to TNF-α in vivo. These findings indicate that CK2 is a key regulator of leukocyte–endothelial cell interaction in inflammation by regulating the expression of E-selectin, ICAM-1 and VCAM-1 via affecting the transcriptional activity of the NF-κB complex. Accordingly, CK2 represents a promising target for the development of novel anti-inflammatory drugs.

Fibrogenesis, novel lessons from animal models.

Systemic sclerosis (SSc) is a devastating chronic autoimmune connective tissue disease characterized by vasculopathy, autoimmunity with inflammation, and progressive fibrogenesis. The current paradigm of the pathogenesis of SSc is that of an unknown initial trigger, leading to a complex interaction of immune cells, endothelial cells, and fibroblasts, producing cytokines, growth and angiogenic factors, and resulting in uncontrolled and persistent tissue fibrogenesis by an altered mesenchymal cell compartment. Animal models are of utmost importance to investigate the different steps in the pathogenesis. This review will elaborate on recent findings in established and more recently developed animal models, presenting data on compounds that are in or ready to be translated into clinical trials, or provide interesting new findings in the understanding of the pathophysiology of SSc. We focus on recent findings concerning the vessel-extracellular matrix interaction, the initial triggering aggressor, the concept of autoimmunity and inflammatory changes, the effector cells and their origins, and the complex interaction of the different signaling pathways in fibrogenesis.

Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.

The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.

Therapeutic effect of methotrexate encapsulated in cationic liposomes (EndoMTX) in comparison to free methotrexate in an antigen-induced arthritis study in vivo

Objectives: Cationic lipid complexes bind to angiogenic endothelial cells of solid tumours and microvessels of chronic inflammatory tissue. Methotrexate (MTX) is one of the drugs used in the therapy of rheumatoid arthritis (RA); it is applied systemically but can have serious side-effects. The aim of this study was to investigate the impact of MTX encapsulated in cationic liposomes (EndoMTX) in comparison to treatment with free MTX.Method: We used an antigen-induced arthritis (AiA) model and investigated the leucocyte– and platelet–endothelial cell interaction in arthritic female C57/Bl6 mice and in healthy controls. The arthritic animals were divided into four different groups receiving either trehalose, free MTX, EndoMTX placebo, or EndoMTX. These parameters and functional capillary density (FCD) were measured and assessed by intravital microscopy (IVM). We controlled clinical parameters such as the knee joint diameter (KJD) throughout the observation period.Results: Animals treated with EndoMTX showed a significant and superior reduction in leucocyte– and platelet–endothelial cell interaction, FCD, and KJD. Free MTX or empty liposomes also showed a reduction in these parameters but not to a significant level. FCD decreased in the EndoMTX group in comparison to using free drugs or empty carrier-like liposomes.Conclusions: This study demonstrates the advantage of using MTX encapsulated in cationic liposomes in contrast to free and generic MTX, with a higher efficacy in anti-inflammatory and anti-angiogenic abilities. Targeting with cationic liposomes may be a promising treatment option and should be elucidated in further experiments regarding dose reduction and side-effects due to MTX usage.

The effect of saffron & its bioactive compound; crocin against monocyte – Endothelial cell interaction in human coronary arterial endothelial cells

The effect of saffron & its bioactive compound; crocin against monocyte – Endothelial cell interaction in human coronary arterial endothelial cells

Effect of Monocyte-Like THP-1 Cells on the Formation of Vascular Tubes by EA.Hy926s Endothelial Cells in the Presence of Cytokines.

The interaction of endothelial cells with cells of the microenvironment, including monocytes/ macrophages, and extracellular matrix during angiogenesis is controlled by cytokines. The stimulating effect bFGF, IL-8, and VEGF on the formation of capillary-like structures by endothelial cells was demonstrated in both monoculture and in co-culture with THP-1 cells; in the latter case, the effects of bFGF and VEGF were more pronounced. IL-8 reduced branching of vascular tubes in co-culture in comparison with monoculture of endothelial cells. Placental growth factor PlGF had no effect of tube formation by endothelial cells in monoculture, but in co-culture with THP-1 cells this cytokine in high concentrations exhibited proangiogenic activity. TGFb inhibited the formation of vascular tubes by endothelial cells and its antiangiogenic potential was more pronounced in co-culture with THP-1 cells.