Relaxin inhibits early steps in vascular inflammation

Increased expression of endothelial adhesion molecules, high levels of the monocyte chemoattractant protein-1 (MCP-1) and enhanced VLA4 integrin/VCAM-1 and CCR-2/MCP-1 interactions are initial steps in vascular inflammation. We sought to determine whether relaxin, a potent vasodilatory and anti-fibrotic agent, mitigates these early events compromising endothelial integrity. The effect of relaxin coincubation on the TNF-α-stimulated expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin; the MCP-1 expression by human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HAoSMC); as well as on direct monocyte–endothelium cell adhesion was quantified by ELISA or adhesion assay. CCR-2 and PECAM expression on HUVEC and THP-1 monocytes was investigated by FACS analysis. Relaxin treatment suppressed significantly TNF-α-induced upregulation of VCAM-1 and PECAM, CCR-2, and MCP-1 levels and direct monocyte adhesion to HUVEC. Our findings identify relaxin as a promising inhibitory factor in early vascular inflammation. By attenuating the upregulation of VCAM-1, key adhesion molecule in early vascular inflammation, and of MCP-1, a chemokine pivotal to monocyte recruitment, relaxin decreased initial monocyte–endothelium contact. This may be of relevance for the prevention and treatment of atherosclerosis and of other pro-inflammatory states.     

Leukocyte – endothelial interactions in inflammation

At sites of inflammation, infection or vascular injury local proinflammatory or pathogen-derived stimuli render the luminal vascular endothelial surface attractive for leukocytes. This innate immunity response consists of a well-defined and regulated multi-step cascade involving consecutive steps of adhesive interactions between the leukocytes and the endothelium. During the initial contact with the activated endothelium leukocytes roll along the endothelium via a loose bond which is mediated by selectins. Subsequently, leukocytes are activated by chemokines presented on the luminal endothelial surface, which results in the activation of leukocyte integrins and the firm leukocyte arrest on the endothelium. After their firm adhesion, leukocytes make use of two transmigration processes to pass the endothelial barrier, the transcellular route through the endothelial cell body or the paracellular route through the endothelial junctions. In addition, further circulating cells, such as platelets arrive early at sites of inflammation contributing to both coagulation and to the immune response in parts by facilitating leukocyte–endothelial interactions. Platelets have thereby been implicated in several inflammatory pathologies. This review summarizes the major mechanisms and molecules involved in leukocyte–endothelial and leukocyte-platelet interactions in inflammation.     

Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

Serum retinol-binding protein 4 (RBP4) is the sole specific vitamin A (retinol) transporter in blood. Elevation of serum RBP4 in patients has been linked to cardiovascular disease and diabetic retinopathy. However, the significance of RBP4 elevation in the pathogenesis of these vascular diseases is unknown. Here we show that RBP4 induces inflammation in primary human retinal capillary endothelial cells (HRCEC) and human umbilical vein endothelial cells (HUVEC) by stimulating expression of proinflammatory molecules involved in leukocyte recruitment and adherence to endothelium, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). We demonstrate that these novel effects of RBP4 are independent of retinol and the RBP4 membrane receptor STRA6 and occur in part via activation of NADPH oxidase and NF-κB. Importantly, retinol-free RBP4 (apo-RBP4) was as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory molecules in both HRCEC and HUVEC. These studies reveal that RBP4 elevation can directly contribute to endothelial inflammation and therefore may play a causative role in the development or progression of vascular inflammation during cardiovascular disease and microvascular complications of diabetes.     

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19.     

Persistent Infection of Human Fetal Endothelial Cells with Rubella Virus

Cardiovascular abnormalities are the leading cause of neonatal death among patients with congenital rubella syndrome (CRS). Although persistence of rubella virus (RV) in fetal endothelium has been repeatedly suggested as a possible cause of cardiovascular birth defects, evidence of the permissiveness of fetal endothelial cells to RV is lacking. In this study we evaluated the ability of RV to infect and persist in primary fetal endothelial cells derived from human umbilical vein (HUVEC). We found that wild type (wt) low passage clinical RV productively infected HUVEC cultures without producing cytopathology or ultrastructural changes. RV did not inhibit host cell protein synthesis, cell proliferation, or interfere with the cell cycle. Persistently infected cultures were easily established at low and high multiplicities of infection (MOI) with both laboratory and wt clinical RV strains. However, synchronous infections of entire HUVEC monolayers were only observed with clinical RV strains. The release of infectious virions into media remained at consistently high levels for several subcultures of infected HUVEC. The results indicate that macrovascular fetal endothelial cells are highly permissive to RV and allow slow persistent RV replication. The findings provide more evidence for the suggestion that vascular pathologies in CRS are triggered by persistent rubella virus infection of the endothelium.     

Bestrophin 3 Ameliorates TNFα-Induced Inflammation by Inhibiting NF-κB Activation in Endothelial Cells

Increasing evidences have suggested vascular endothelial inflammatory processes are the initiator of atherosclerosis. Bestrophin 3 (Best-3) is involved in the regulation of cell proliferation, apoptosis and differentiation of a variety of physiological functions, but its function in cardiovascular system remains unclear. In this study, we investigated the effect of Best-3 on endothelial inflammation. We first demonstrated that Best-3 is expressed in endothelial cells and decreased after tumor necrosis factor-α (TNFα) challenge. Overexpression of Best-3 significantly attenuated TNFα-induced expression of adhesion molecules and chemokines, and subsequently inhibited the adhesion of monocytes to human umbilical vein endothelial cells (HUVECs). Conversely, knockdown of Best-3 with siRNA resulted in an enhancement on TNFα-induced expression of adhesion molecules and chemokines and adhesion of monocytes to HUVECs. Furthermore, overexpression of Best-3 with adenovirus dramatically ameliorated inflammatory response in TNFα-injected mice. Mechanistically, we found up-regulation of Best-3 inhibited TNFα-induced IKKβ and IκBα phosphorylation, IκBα degradation and NF-κB translocation. Our results demonstrated that Best-3 is an endogenous inhibitor of NF-κB signaling pathway in endothelial cells, suggesting that forced Best-3 expression may be a novel approach for the treatment of vascular inflammatory diseases.     

Toxic effects of interleukin-2-activated lymphocytes on vascular endothelial cells

IL-2-activated lymphocytes (LAK cells) show increased adherence to, and killing of, human vascular endothelial cells compared to resting lymphocytes. In the present work, we have found that supernatants from LAK cell cultures also are toxic to human umbilical vein endothelial cells (HUVEC) when tested for 48 h in a neutral red uptake assay. Recombinant TNF-alpha and IFN-gamma at high concentrations are also toxic under the same test conditions, and TNF-alpha was directly detected in LAK cell supernatants. An inconsistent inhibition of toxicity was found with anti-TNF-alpha whereas anti IFN-gamma antibodies had a partial inhibitory effect. The susceptibility of HUVEC to cellular killing by LAK cells could be up- and down-regulated with insulin-like growth factor I and IFN-gamma, respectively. It is concluded that damage to vascular endothelium during high dose IL-2 treatments may be partially related to an excessive production of lymphokines such as IFN-gamma and TNF-alpha. IFN-gamma may, in addition, be protective for HUVEC during cellular interactions with LAK cells.     

The molecular and physiological aspects of endothelial dysfunction. The role of endogenous chemical regulators

Monolayer of endothelial cells that cover the vascular channels are the major regulator of haemo-vascular homeostasis. Endothelium secretes the chemical factors that affect contraction of the muscular vascular cells, permeability of tissue, blood fluidity, intercellular interaction in vascular structure of the channel as a whole and of different regions. In its turn, the secretory function of endothelial cells is stimulated by mechanical or hormonal factors under a feedback system principle. Special features of morphology and biochemistry of vascular endothelium cells determine the micro-organs heterogeneity of the vascular channel depending on phenotine, gene expression, size and growth of endothelium cells. On this basis the processing biochemical disintegration develop either selectively or in a generalised form, and results in development of endothelial dysfunctions, as the original factor of many cardiovascular pathologies. Endothelial disfunction is a systemic pathology related to pathology of microstructure and hormonal function of endothelial cells representing a major tissue system of the vascular channel. Formation of hypertension states, ischemic cardiopathology, haemostasis changes, metabolic pathology (hypercholesterinemia and hyperglycemia) that lead to pathogenesis of arteriosclerosis, diabetes (etc.) as result of modified function of endothelium, and above all, pathology of production by dilator and constrictor substances, and the factors regulating interaction of endothelium with blood cells. The basic mechanism for development of the endothelial dysfunction is related to modification of synthesis and releasing of nitrogen oxide, a key regulator of the endothelial-vasal system. Physiologically active peptides (angiotensin II, endothelin-I, bradykinin, adrenomedullin and ANP) contribute to development of the processes related to the endothelium function and dysfunction. An important role is played, apparently, by growth peptide factors and specific proteins of cellular adhesion and membrane interaction--to integrins and selectins.     

Proinflammatory response of human endothelial cells to Brucella infection

Although vascular pathologies such as vasculitis, endocarditis and mycotic aneurysms have been described in brucellosis patients, the interaction of Brucella with the endothelium has not been characterized. In this study we show that Brucella abortus and Brucella suis can infect and replicate in primary human umbilical vein endothelial cells (HUVEC) and in the microvascular endothelial cell line HMEC-1. Infection led to an increased production of IL-8, MCP-1 and IL-6 in HUVEC and HMEC-1 cells, and an increased expression of adhesion molecules (CD54 in both cells, CD106 and CD62E in HUVEC). Experiments with purified antigens from the bacterial outer membrane revealed that lipoproteins (Omp19) but not lipopolysaccharide mediate these proinflammatory responses. Infection of polarized HMEC-1 cells resulted in an increased capacity of these cells to promote the transmigration of neutrophils from the apical to the basolateral side of the monolayer, and the same phenomenon was observed when the cells were stimulated with live bacteria from the basolateral side. Overall, these results suggest that Brucella spp. can infect and survive within endothelial cells, and can induce a proinflammatory response that might be involved in the vascular manifestations of brucellosis.     

Mechanisms of selective leukocyte recruitment from whole blood on cytokine-activated endothelial cells under flow conditions

Selective recruitment of eosinophils to sites of allergic and parasitic inflammation involves specific adhesion and activation signals expressed on or presented by stimulated endothelial cells. Here we examined leukocyte recruitment on cytokine-activated HUVEC under flow conditions. We perfused whole blood through a flow chamber to examine mechanisms of selective leukocyte recruitment. Although there was substantial recruitment of leukocytes on TNF-alpha-stimulated HUVEC, we found no selective accumulation of any particular leukocyte subpopulations. In contrast, fewer leukocytes were recruited to IL-4-stimulated HUVEC, but the recruitment was selective for eosinophils. We examined the role of adhesion molecules in these interactions and found that eosinophil recruitment was completely blocked with an alpha4 integrin mAb at the shear rates examined. A significant number of neutrophils were also recruited to IL-4-stimulated HUVEC, and these interactions required P-selectin and P-selectin glycoprotein ligand-1. Thus, whole blood perfusion over cytokine-activated endothelium revealed that IL-4-stimulated HUVEC support selective recruitment of eosinophils, whereas TNF-alpha-stimulated HUVEC lack selectivity for any leukocyte subclass.     

TNFα Signals via p66Shc to Induce E-Selectin,Promote Leukocyte Transmigration and Enhance Permeability in Human Endothelial Cells

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66^Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66^Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66^Shc on Ser³⁶ and the stress kinase c-Jun NH₂-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66^Shc in HUVEC resulted in enhanced p66^Shc phosphorylation on Ser³⁶, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66^Shc protein, in which Ser³⁶ was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66^Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66^Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66^Shc on Ser³⁶.     

Glucagon-Like Peptide-1 (GLP-1) Analog Liraglutide Inhibits Endothelial Cell Inflammation through a Calcium and AMPK Dependent Mechanism

Liraglutide is a glucagon-like peptide-1 (GLP-1) mimetic used for the treatment of Type 2 diabetes. Similar to the actions of endogenous GLP-1, liraglutide potentiates the post-prandial release of insulin, inhibits glucagon release and increases satiety. Recent epidemiological studies and clinical trials have suggested that treatment with GLP-1 mimetics may also diminish the risk of cardiovascular disease in diabetic patients. The mechanism responsible for this effect has yet to be determined; however, one possibility is that they might do so by a direct effect on vascular endothelium. Since low grade inflammation of the endothelium is an early event in the pathogenesis of atherosclerotic cardiovascular disease (ASCVD), we determined the effects of liraglutide on inflammation in cultured human aortic endothelial cells (HAECs). Liraglutide reduced the inflammatory responses to TNFα and LPS stimulation, as evidenced by both reduced protein expression of the adhesion molecules VCAM-1 and E-Selectin, and THP-1 monocyte adhesion. This was found to result from increased cell Ca²⁺ and several molecules sensitive to Ca²⁺ with known anti inflammatory actions in endothelial cells, including CaMKKβ, CaMKI, AMPK, eNOS and CREB. Treatment of the cells with STO-609, a CaMKK inhibitor, diminished both the activation of AMPK, CaMKI and the inhibition of TNFα and LPS-induced monocyte adhesion by liraglutide. Likewise, expression of an shRNA against AMPK nullified the anti-inflammatory effects of liraglutide. The results indicate that liraglutide exerts a strong anti-inflammatory effect on HAECs. They also demonstrate that this is due to its ability to increase intracellular Ca²⁺ and activate CAMKKβ, which in turn activates AMPK.     

KRIT1 Protein Depletion Modifies Endothelial Cell Behavior via Increased Vascular Endothelial Growth Factor (VEGF) Signaling

Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1^+/− mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1.     

Incorporation of adult organ-derived endothelial cells into tumor blood vessel

In this study, we attempted to assess the incorporable potential of vascular endothelial cells derived from adult organ blood vessels into tumor blood vessels. Two kinds of adult organ-derived vascular endothelial cells, human aorta endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC), were administered into murine tumors inoculated to SCID mice. Many human blood vessel networks were visualized in the murine tumors. These cells in solid tumor not only survived and proliferated, but also incorporated into tumor endothelium. These results suggest that adult organ-derived vascular endothelial cells possess the potential to form the neovascular network in various tissues such as vascular endothelial progenitor-like cells in vivo. We propose that these cells can be regarded as a congenic (autologous) vector for vascular regeneration cell therapy and tumor vascular targeting gene therapy.     

A Novel Technique for Culture of Human Dermal Microvascular Endothelial Cells under either Serum-Free or Serum-Supplemented Conditions: Isolation by Panning and Stimulation with Vascular Endothelial Growth Factor

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptorkdr,since mRNA forkdrwas detected using RT–PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.     

Induction of Endothelial Cell Apoptosis by Solid Tumor Cells

The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performedin vivoandin vitrodemonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.     

Hydroxycarbamide Decreases Sickle Reticulocyte Adhesion to Resting Endothelium by Inhibiting Endothelial Lutheran/Basal Cell Adhesion Molecule (Lu/BCAM) through Phosphodiesterase 4A Activation

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α₄β₁ integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.     

Endothelial progenitor cells induce a phenotype shift in differentiated endothelial cells towards PDGF/PDGFRβ axis-mediated angiogenesis

Background

Endothelial Progenitor Cells (EPC) support neovascularization and regeneration of injured endothelium both by providing a proliferative cell pool capable of differentiation into mature vascular endothelial cells and by secretion of angiogenic growth factors.

Objective

The aim of this study was to investigate the role of PDGF-BB and PDGFRβ in EPC-mediated angiogenesis of differentiated endothelial cells.

Methods and Results

Conditioned medium from human EPC (EPC-CM) cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01). EPC-CM increased proliferation (1.39-fold; P<0.001) and migration (2.13-fold; P<0.001) of isolated human umbilical vein endothelial cells (HUVEC), as well as sprouting of vascular structures from ex vivo cultured aortic rings (2.78-fold increase; P = 0.01). The capacity of EPC-CM to modulate the PDGFRβ expression in HUVEC was assessed by western blot and RT-PCR. All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFRβ (P<0.01). EPC-CM triggered a distinct up-regulation of PDGFRβ (2.5±0.5; P<0.05) and its phosphorylation (3.6±0.6; P<0.05) in HUVEC. This was not observed after exposure of HUVEC to recombinant human PDGF-BB alone.

Conclusion

These data indicate that EPC-CM sensitize endothelial cells and induce a pro-angiogenic phenotype including the up-regulation of PDGFRβ, thereby turning the PDGF/PDGFRβ signaling-axis into a critical element of EPC-induced endothelial angiogenesis. This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.     

Annexin Peptide Ac2-26 Suppresses TNFα-Induced Inflammatory Responses via Inhibition of Rac1-Dependent NADPH Oxidase in Human Endothelial Cells

The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNFα) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2′,7′-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NFκB) was measured by luciferase activity assay. TNFα stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5–1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNFα-induced NFκB promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNFα whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNFα-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNFα-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNFα-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NFκB pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.