Maintenance Of Vascular Integrity Research Articles

Blockade of PDE4B limits lung vascular permeability and lung inflammation in LPS-induced acute lung injury

Acute lung injury (ALI), acute respiratory distress syndrome (ARDS), is actually involved in an ongoing and uncontrolled inflammatory response in lung tissues. Although extensive studies suggested that phospodiesterase type 4B (PDE4B) may be related to inflammation, the underlying cell biological mechanism of ALI remains unclear. To further investigate the mechanism how PDE4B take part in inflammatory response and the maintenance of vascular integrity, we established the experimental model of ALI in vitro and in vivo. In vitro, we found that Cilomilast, Diazepam and PDE4B knockout could potently inhibit the LPS-induced NF-κB activation and inflammatory response in multiple cell types, including lung epithelial cells (A549), pulmonary microvascular endothelial cells (PMVECs) and vascular smooth muscle cells (VSMCs). Besides, PDE4B deletion attenuated the LPS-induced ROS generation. In vivo, PDE4B deletion could attenuate the lung water content, histological signs of pulmonary injury and elevate the ratio of partial pressure of arterial O2 to fraction of inspired O2 (PaO2/FIO2 ratio). Additionally, PDE4B deletion reduced LPS-induced vascular permeability. Collectively, our results strongly indicates that PDE4B is a valid target for anti-ALI.

Angiogenesis effect of Astragalus polysaccharide combined with endothelial progenitor cells therapy in diabetic male rat following experimental hind limb ischemia

Background Diabetes mellitus (DM) is a common disease accompanied with a high incidence of hind limb ischemia (HLI). In recent years, numerous studies demonstrated that endothelial progenitor cells (EPCs) are involved in angiogenesis and maintenance of vascular integrity following HLI. On the other side, it has been proved that Astragalus polysaccharide (APS) could promote angiogenesis. In the present study, we aimed to evaluate the effect of APS and EPCs on enhancing angiogenesis after experimental HLI caused by femoral artery ligation in rats with streptozotocin (STZ)-induced diabetes. Methods Rats (n=110) were randomly assigned to the following groups: sham group, ischemia group, APS group, EPCs group and APS+EPCs group. APS, EPCs or an equal volume of vehicle was administered intramuscularly after HLI induction, and 6 rats were assessed by angiography at 28 days after induction of HLI, 6 rats were sacrificed at the same time point to take histological studies, biochemical tests were also performed at that point in the rest rats. Results APS or EPCs treatment induced an increase, respectively, in the protein expression of vascular endothelial growth factor (VEGF) (36.61%, 61.59%), VEGF receptor-1 (VEGFR-1) (35.50%, 57.33%), VEGFR-2 (31.75%, 41.89%), Angiopoietin-1 (Ang-1) (37.57%, 64.66%) and Tie-2 (42.55%, 76.94%) (P <0.05), after HLI injury. And combined therapy of APS and EPCs enhanced the effort of angiogenesis after HLI induction in diabetic rats, through elevating protein expression of VEGF (99.67%), VEGFR-1 (105.33%), VEGFR2 (72.05%), Ang-1 (114.30%) and Tie-2 (111.87%) (P<0.05). Similarly, mRNA expression of VEGF, VEGFR-1, VEGFR2, Ang-1, Tie-2 also show similar trends as well as protein expression (P<0.05). Conclusion APS or EPCs could enhance angiogenesis, and the combined treatment leads to better effort, at least, partially via VEGF/VEGFR and Ang-1/Tie-2 signaling pathway.

MiRNAs in peripheral artery disease - something gripping this way comes.

Peripheral artery disease (PAD) is a marker disease for generalized atherosclerosis and represents one of the world's major causes of morbidity and mortality. Many studies have tried to gain insight into the molecular mechanisms involved in PAD onset, progression and prognosis. In the last decade, small non-coding RNAs, termed miRNAs, have emerged as a major research focus due to their regulating function of multiple gene sets. In the cardiovascular system, miRNAs not only impact on physiological pathways like cardiac development and angiogenesis, but also play an important role in disease mechanisms and progression of myocardial hypertrophy, acute myocardial infarction, heart failure or arrhythmias. New insights lend considerable support to the concept of miRNAs serving as highly sensitive biomarkers and therapeutic targets. To date, a comprehensive understanding of miRNA regulation of angiogenesis and maintenance of vascular integrity in PAD remains less explored. In this review, we discuss current studies and highlight the potential of miRNAs not only to act as a diagnostic tool, but also to facilitate innovative strategies for gene therapy.

Renal Ischemia-Reperfusion Induces Release of Angiopoietin-2 From Human Grafts of Living and Deceased Donors

Recent insights suggest that endothelial cell (EC) activation plays a major role in renal ischemia-reperfusion (I/R) injury. Interactions between ECs and pericytes via signaling molecules, including angiopoietins, are involved in maintenance of the vascular integrity. Experimental data have shown that enhancement of Angiopoietin (Ang)-1 signaling might be beneficial in renal I/R injury. However, little is known about the role of angiopoietins in human renal I/R injury. In this study, EC activation and changes in angiopoeitins are assessed in human living-donor (LD) and deceased-donor (DD) kidney transplantation. Local release of angiopoietins was measured by unique, dynamic arteriovenous measurements over the reperfused kidney. Renal I/R is associated with acute EC activation shown by a vast Ang-2 release from both LD and DD shortly after reperfusion. Its counterpart Ang-1 was not released. Histologic analysis of kidney biopsies showed EC loss after reperfusion. Baseline protein and mRNA Ang-1 expression was significantly reduced in DD compared with LD and declined further after reperfusion. Human renal I/R injury induces EC activation after reperfusion reflected by Ang-2 release from the kidney. Interventions aimed at maintenance of vascular integrity by modulating angiopoietin signaling may be promising in human clinical kidney transplantation.

Abstract 486: Platelet ITAM Signaling is Critical for Maintenance of Vascular Integrity in Inflammation

Blood platelets, long recognized for their importance in primary hemostasis, have recently been identified as critical regulators of vascular integrity during inflammation and cancer. Interestingly, the contribution of platelets to the maintenance of vascular integrity in in[[Unable to Display Character: fl]]ammation seems to be independent of their ability to form a clot. However, the underlying molecular mechanism remains unknown. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling but not G protein-coupled receptor (GPCR) signaling is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a novel protocol for the adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from wild-type platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of CLEC-2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into thrombocytopenic mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel anti-thrombotic agents directed towards the ITAM signaling pathway.

Ve-ptp Modulates Vascular Integrity by Promoting Adherens Junction Maturation

BackgroundEndothelial cell junctions control blood vessel permeability. Altered permeability can be associated with vascular fragility that leads to vessel weakness and haemorrhage formation. In vivo studies on the function of genes involved in the maintenance of vascular integrity are essential to better understand the molecular basis of diseases linked to permeability defects. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein present at endothelial adherens junctions (AJs).Methodology/Principal FindingsWe investigated the role of Ve-ptp in AJ maturation/stability and in the modulation of endothelial permeability using zebrafish (Danio rerio). Whole-mount in situ hybridizations revealed zve-ptp expression exclusively in the developing vascular system. Generation of altered zve-ptp transcripts, induced separately by two different splicing morpholinos, resulted in permeability defects closely linked to vascular wall fragility. The ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos.Conclusions/SignificanceHere we show the first in vivo evidence of a potentially critical role played by Ve-ptp in AJ maturation, an important event for permeability modulation and for the development of a functional vascular system.

Fibroblast Growth Factor Signaling Potentiates VE-Cadherin Stability at Adherens Junctions by Regulating SHP2

BackgroundThe fibroblast growth factor (FGF) system plays a critical role in the maintenance of vascular integrity via enhancing the stability of VE-cadherin at adherens junctions. However, the precise molecular mechanism is not well understood. In the present study, we aimed to investigate the detailed mechanism of FGF regulation of VE-cadherin function that leads to endothelial junction stabilization.Methods and FindingsIn vitro studies demonstrated that the loss of FGF signaling disrupts the VE-cadherin-catenin complex at adherens junctions by increasing tyrosine phosphorylation levels of VE-cadherin. Among protein tyrosine phosphatases (PTPs) known to be involved in the maintenance of the VE-cadherin complex, suppression of FGF signaling reduces SHP2 expression levels and SHP2/VE-cadherin interaction due to accelerated SHP2 protein degradation. Increased endothelial permeability caused by FGF signaling inhibition was rescued by SHP2 overexpression, indicating the critical role of SHP2 in the maintenance of endothelial junction integrity.ConclusionsThese results identify FGF-dependent maintenance of SHP2 as an important new mechanism controlling the extent of VE-cadherin tyrosine phosphorylation, thereby regulating its presence in adherens junctions and endothelial permeability.

Impact of oral simvastatin therapy on acute lung injury in mice during pneumococcal pneumonia

BackgroundRecent studies suggest that the reported protective effects of statins (HMG-CoA reductase inhibitors) against community-acquired pneumonia (CAP) and sepsis in humans may be due to confounders and a healthy user-effect. To directly test whether statins are protective against Streptococcus pneumoniae, the leading cause of CAP, we examined the impact of prolonged oral simvastatin therapy at physiologically relevant doses in a mouse model of pneumococcal pneumonia. BALB/c mice were placed on rodent chow containing 0 mg/kg (control), 12 mg/kg (low simvastatin diet [LSD]; corresponds to 1.0 mg/kg/day), or 120 mg/kg (high simvastatin diet [HSD]; corresponds to 10 mg/kg/day) simvastatin for four weeks, infected intratracheally with S. pneumoniae serotype 4 strain TIGR4, and sacrificed at 24, 36, or 42 h post-infection for assessment of lung histology, cytokine production, vascular leakage and edema, bacterial burden and bloodstream dissemination. Some mice received ampicillin at 12-h intervals beginning at 48 h post-infection and were monitored for survival. Immunoblots of homogenized lung samples was used to assess ICAM-1 production.ResultsMice receiving HSD had reduced lung consolidation characterized by less macrophage and neutrophil infiltration and a significant reduction in the chemokines MCP-1 (P = 0.03) and KC (P = 0.02) and ICAM-1 in the lungs compared to control mice. HSD mice also had significantly lower bacterial titers in the blood at 36 (P = 0.007) and 42 (P = 0.03) hours post-infection versus controls. LSD had a more modest effect against S. pneumoniae but also resulted in reduced bacterial titers in the lungs and blood of mice after 42 h and a reduced number of infiltrated neutrophils. Neither LSD nor HSD mice had reduced mortality in a pneumonia model where mice received ampicillin 48 h after challenge.ConclusionsProlonged oral simvastatin therapy had a strong dose-dependent effect on protection against S. pneumoniae as evidenced by reduced neutrophil infiltration, maintenance of vascular integrity, and lowered chemokine production in the lungs of mice on HSD. Statin therapy also protected through reduced bacterial burden in the lungs. Despite these protective correlates, mortality in the simvastatin-receiving cohorts was equivalent to controls. Thus, oral simvastatin at physiologically relevant doses only modestly protects against pneumococcal pneumonia.

Abstract 53: Immunoreceptor Tyrosine Activation Motif Signaling in Platelets Is Critical for the Maintenance of Vascular Integrity During Inflammation

Introduction: Blood platelets, long recognized for their importance in primary hemostasis, have recently been identified as critical regulators of vascular integrity during inflammation and cancer. Interestingly, the contribution of platelets to the maintenance of vascular integrity in inflammation seems to be independent of their ability to form a clot. Goal: To identify signaling molecules critical for the contribution of platelets to vascular integrity in inflammation. Methods: Transgenic mice with platelet-specific expression of the interleukin (IL)4 receptor were rendered thrombocytopenic by infusion of antibodies to IL4R. Platelets from various knockout mice or inhibitor-treated wild-type (WT) platelets were transfused into thrombocytopenic (TP) mice and tested for their ability to support vascular integrity in a reverse passive Arthus reaction (rpA) model. Tissue hemoglobin (Hb) was used to quantify red blood cells in the inflamed tissue, a marker of disrupted vascular integrity. Results: Impaired vascular integrity at the site of inflammation was markedly higher in TP mice compared to controls (Hb: 1448±176 vs. 100±8 μg/cm2). Transfusion of WT platelets into TP mice strongly reduced tissue Hb (150±46 μg/ cm2). Transfused platelets from mice lacking the thrombin receptor PAR4 and/or functional P2Y12 (ADP receptor) also prevented disruption of vascular integrity (Hb: 210±66, 136±16 μg and 360±120 μg/cm2), suggesting that signaling by G protein-coupled receptors is not important for this response. Furthermore, infusion of heparin into WT mice did not lead to increased tissue Hb. Immunoreceptor tyrosine activation motif (ITAM) signaling, however, was critical for platelet-dependent maintenance of vascular integrity, as tissue Hb was markedly increased in TP mice transfused with platelets lacking the signal trandsucing adapter protein SLP-76 (1212±160 μg/cm2) or platelets lacking functional ITAM signaling receptors GPVI (852±196 μg/cm2), CLEC-2 (1062±188 μg/cm2), or both (1498±128 μg/cm2). Data are mean±SEM, n=4-5 mice per group. Conclusions: We have established a novel mouse model for the adoptive transfer of platelets into TP mice. Using this model we can show that the ITAM but not GPCR signaling in platelets is critical for the maintenance of vascular integrity during inflammation. Our studies highlight a potential clinical complication of future anti-platelet agents targeted towards ITAM signaling receptors such as GPVI.

Toe Tissue Transfer for Reconstruction of Damaged Digits due to Electrical Burns

BackgroundElectrical burns are one of the most devastating types of injuries, and can be characterized by the conduction of electric current through the deeper soft tissue such as vessels, nerves, muscles, and bones. For that reason, the extent of an electric burn is very frequently underestimated on initial impression.MethodsFrom July 1999 to June 2006, we performed 15 cases of toe tissue transfer for the reconstruction of finger defects caused by electrical burns. We performed preoperative range of motion exercise, early excision, and coverage of the digital defect with toe tissue transfer.ResultsWe obtained satisfactory results in both functional and aesthetic aspects in all 15 cases without specific complications. Static two-point discrimination results in the transferred toe cases ranged from 8 to 11 mm, with an average of 9.5 mm. The mean range of motion of the transferred toe was 20° to 36° in the distal interphalangeal joint, 16° to 45° in the proximal interphalangeal joint, and 15° to 35° in the metacarpophalangeal joint. All of the patients were relatively satisfied with the function and appearance of their new digits.ConclusionsThe strategic management of electrical injury to the hands can be both challenging and complex. Because the optimal surgical method is free tissue transfer, maintenance of vascular integrity among various physiological changes works as a determining factor for the postoperative outcome following the reconstruction.

Impaired Colony-Forming Capacity of Circulating Endothelial Progenitor Cells in Patients with Emphysema

Chronic obstructive pulmonary disease (COPD) is classified into emphysema and chronic bronchitis, which are thought to result from different pathophysiological pathways. Smoking-induced lung parenchymal destruction and inadequate repair are involved in the pathogenesis of emphysema. In addition, decreased expression of vascular endothelial growth factor and increased endothelial cell apoptosis in the lung may participate in emphysema pathogenesis. As stem cells, circulating endothelial progenitor cells (EPCs) may play a key role in the maintenance of vascular integrity by replacing and repairing the damaged endothelial cells in the tissues. To determine whether the lack of appropriate repair by circulating EPCs in cases of smoking-induced endothelial cell injury participates in emphysema pathogenesis, we determined the association between the colony-forming or migratory capacity of circulating EPCs and the presence of emphysema in 51 patients with COPD. The patients were divided into emphysema (n = 23) and non-emphysema groups (n = 28) based on high-resolution computed tomography. Twenty-two smokers with normal lung function and 14 normal non-smokers served as controls. Circulating EPCs isolated from patients with emphysema showed significantly lower colony-forming units (CFUs) than those from patients with non-emphysema group, smokers with normal lung function, and normal non-smokers. EPCs from patients with emphysema showed significantly lower migratory capacity than those from normal non-smoking controls (p < 0.05). On multivariate analysis, the EPC-CFU was independently associated with emphysema (OR 0.944, 95% CI = 0.903-0.987, p = 0.011). Thus, impaired functions of circulating EPCs may contribute to the development of emphysema.

Thrombin Supports Colitis and Colitis-Associated Cancer Pathogenesis

Abstract 2237Chronic inflammation has been recognized as a major factor in the development and progression of multiple cancers. A prime example of this is the strong association between colitis and colon cancer. However, the specific factors that regulate disadvantageous immune processes in the context of inflammation-associated cancers remain poorly defined. Growing evidence suggests that hemostatic system components, traditionally associated with the maintenance of vascular integrity and prevention of blood loss, also directly regulate inflammatory processes. Furthermore, thrombin and several thrombin targets (e.g., PARs, fibrinogen, factor XIII) have been shown to regulate tumor cell proliferation and apoptosis, support metastasis, and protect tumor cells from innate immune surveillance mechanisms in other experimental contexts. A logical extension of these findings is the hypothesis that thrombin, as a master regulator of both inflammatory processes and tumor cell biology, is a major determinant of the progression of inflammation-driven cancers such as colitis-associated colon cancer (CAC). To test this hypothesis, we induced CAC in mice carrying prothrombin levels 50% of normal (fII+/−) and WT mice in parallel using an established two step protocol consisting of azoxymethane (AOM) and dextran sodium sulfate (DSS) exposure. The modest diminution in prothrombin levels imposed by the fII+/− genotype resulted in a dramatic diminution in the number of colonic adenomas formed after AOM/DSS challenge relative to WT mice. In order to determine if the diminution in adenoma formation observed in fII+/− mice was coupled to thrombin function, wildtype mice challenged with AOM/DSS were treated with daily i.p. injections of hirudin, a direct thrombin inhibitor, or saline carrier. Similar to the finding in mice with a genetically-imposed diminution in circulating prothrombin, hirudin treatment significantly blunted adenoma formation. To determine if reduction of thrombin generation improved the inflammation preceding the development of colonic adenomas, we used a novel, highly-specific factor XI antisense oligonucleotide “gapmer” (ISIS Pharmaceuticals) to inhibit hepatic factor XI synthesis prior to DSS challenge. Gapmer-mediated diminution of fXI levels to ∼15% of normal resulted in a dramatic improvement in colitis related symptoms. Gapmer-treated mice had less intestinal bleeding and weight loss associated with DSS challenge relative to mice treated with a control oligonucleotide. Consistent with these gross observations, microscopic analyses of colonic tissue showed that fXI gapmer treatment significantly limited mucosal ulceration. Factor XI gapmer treatment also significantly diminished local levels of several inflammatory cytokines known to play a role in colon cancer progression (i.e., IL-6, IL-1β, IL-12). These results demonstrate that thrombin is a crucial driver of the pathogenesis of colitis-associated colon cancer and suggest that therapies directed at thrombin or thrombin generation could treat or prevent inflammation-driven colon cancer. As pathological inflammation has been estimated to account for as many as 1 in 5 cancer-related deaths, thrombin-directed therapies could have broad applicability to multiple malignancies. Disclosures:Mullins:Baxter: Consultancy. Monia:Isis Pharmaceuticals: Employment. MacLeod:Isis Pharmaceuticals: Employment. Revenko:Isis Pharmaceuticals: Employment. Palumbo:Novo Nordisk: Research Funding.

Opposing Roles for CD34 in B16 Melanoma Tumor Growth Alter Early Stage Vasculature and Late Stage Immune Cell Infiltration

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34−/− mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34−/− mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34−/− mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.

FGF‐VEGF crosstalk regulating vascular integrity and angiogenesis

We have recently shown that the maintenance of vascular integrity is an active process which requires basal FGF signaling. This implies interplay of FGF and VEGF in fundamental vascular functions. In fact, numerous studies have suggested a link between FGF and VEGF signaling; however the nature of this crosstalk remains elusive. We assessed the hypothesis that FGF controls vessel growth through regulating VEGF activity. To this end, we investigated VEGF signaling in endothelial cells (EC) with disrupted FGF signaling. We found that EC lacking FGF signaling become unresponsive to VEGF due to downregulation of VEGFR2 expression caused by decreased Vegfr2 enhancer activity. This was resulted from reduced activation of Ets family transcription factors. Erk1/2 activation followed by binding of Ets proteins to the FOX:ETS composite motif in the Vegfr2 enhancer is critical to this regulation. In vivo, lack of endothelial FGF signaling manifested in the loss of vascular integrity and morphogenesis in multiple neovascularization models in mice, and impaired vessel formation caused by FGF inhibition was rescued by exogenous VEGFR2 expression. In conclusion, endothelial FGF signaling controls critical vascular functions via promoting VEGFR2 expression and thereby modifying the response to VEGF. Thus, this crosstalk accounts for the hierarchic regulation of vascular formation by FGF and VEGF. (NIH HL053793)

The Integrin Co-Activator Kindlin-2 Plays a Critical Role In Angiogenesis and Blood Vessel Integrity

AbstractAbstract 4Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Our previous study demonstrated that reduction of kindlin-2 levels in human endothelial cells results in defective adhesive and migratory responses, suggesting that kindlin-2 may be implicated in angiogenesis. We tested this hypothesis in the kindlin-2+/− mice utilizing murine RM1 prostate tumor and Matrigel implant models. Staining of tumor sections for EC with CD31 showed shorter and thinner blood vessels and reduced vascular area by 3.5-fold in tumors grown in kindlin-2+/− mice compared to WT mice (P=0.0186, n=6). The vessels that did form in the kindlin-2+/− mice were immature as they lacked smooth muscle cells and pericytes, had thinner basement membrane, and were leaky as evidenced by increased by 2-fold area for plasma-derived fibrin in tumor sections (P=0.0006, n=5). Consistent with the blunted angiogenic response and vascular leakiness in the kindlin-2+/− mice, the tumors grown in kindlin-2+/− animals had 2-fold larger necrotic core and were 2.5-fold smaller than those derived in WT mice (P=0.042, n=7). Also, the permeability of preexisting blood vessels in ear and dorsal skin of WT and kindlin-2+/− mice was compared after injection of Evans blue dye. Baseline permeability of vasculature in ear and dorsal skin was enhanced by ∼70–100 % in kindlin-2+/− mice as compared WT mice (P<0.01, n=6). Application of mustard oil, a proinflammatory stimulus, on ear skin or VEGF on dorsal skin, enhanced permeability in WT mice by ∼75–80 % (P<0.05, n=6) while it had a negligible effect in the kindlin-2+/− mice (P=0.159, n=6). The impaired angiogenic response in the kindlin-2+/− mice was confirmed in a Matrigel model where VEGF-induced angiogenesis was abnormal. CD31 staining showed that in contrast to the matrigel plugs derived from WT mice, those obtained from the kindlin-2+/− mice contained shorter and thinner vessels, and the few thicker CD31+ structures that did form in the kindlin-2+/− mice were disorganized. As a consequence of blood leakage from abnormal vessels the Matrigel implants collected from the kindlin-2+/− mice exhibited a 3.5-fold increase in hemoglobin content as compared to WT mice (P=0.0149, n=5).Bone marrow transplant experiments were performed and showed that bone marrow-derived cells did not significantly contribute to abnormal angiogenesis and enhanced vascular permeability observed in the kindlin-2+/− mice. Tumor vascular area, plasma-derived fibrin content, tumor growth and vascular permeability in ear and dorsal skin were similar in WT recipients, which received bone marrow from kindlin-2+/− or WT donors. Aortic endothelial cells isolated from WT and kindlin-2+/− mice exhibited similar expression levels of the β1 and β3 integrins as well as VEGFR2. However, FACS analysis of soluble ligand binding showed significantly reduced activation of the β3 integrins in kindlin-2+/− ECs as compared to WT cells, whereas the β1 integrin activation was similar in both genotypes. Additionally, PMA or VEGF enhanced integrin activation in WT ECs, but failed to do so in the kindlin-2+/− cells. Accordingly, integrin-dependent cellular functions: adhesion, migration and spreading of the EC derived from the kindlin-2+/− mice were significantly blunted on the β3 integrin substrates but not on the β1 substrates. In control experiments, overexpression of kindlin-2 in the kindlin-2+/− EC restored defective spreading on vitronectin while a kindlin-2 that does not bind integrins, did not, indicating specificity. Also, tube formation by kindlin-2+/− ECs was significantly impaired compared to those developed by WT ECs. Taken together, kindlin-2 plays an important role in angiogenesis as well as in maintenance of vascular integrity. Disclosures:No relevant conflicts of interest to declare.

Troubleshooting in platelet storage temperature and new perspectives through proteomics.

Platelets represent a key cellular blood component under physiological conditions, due to their implications in the maintenance of vascular integrity and prevention of haemorrhagic phenomena1. Dysfunctions associated with thrombocytopenia constitute a significant threat to patients’ health. So far, administration of platelet concentrates (PCs) from healthy donors is the only known strategy for medical care of patients with active bleeding, thrombocytopenia caused by bone marrow dysfunctions or due to chemotherapy for treatments of malignancies, or upon preliminary treatments prior to stem cell transplantation. Qualitative and quantitative changes in platelets could also occur following coronary artery bypass surgery and trauma, and may represent a key indicator of likely thrombotic complications2. Platelets are basically collected from donors in two ways: extraction from whole blood throughout centrifugation (buffy-coat) or through apheresis. Both methods are currently employed and have bright sides and disadvantages. Analogously to other blood components, techniques to improve the shelf life of platelets (currently, only five days), while maintaining their safety and effectiveness, are under constant investigation worldwide. Indeed, blood transfusion services are always in shortage of PCs, because of the impossibility to prolong their storage in like fashion to hypothermic storage of erythrocyte concentrates or frozen storage of fresh frozen plasma. In order to improve storage of PCs, lowering temperature has been attempted as well, although results were not as positive as expected, since platelets do not tolerate refrigeration. Hypothermic (4°C) storage conditions cause deep modifications in platelet shape and functionality. These are relevant issues which compromise viability of cold stored platelets, as they exert their physiological role through their ability to change shape and activate under various conditions. Low temperature appears to be a triggering factor for activation as well, thus yielding yet activated platelets as unviable blood product at the end of the storage. This review focuses on platelet storage temperatures and the main issues which affect both current (22 °C) and alternative (4 °C) storage protocols for PCs. In particular, we herein discuss the initial events which trigger morphological and physiological changes upon cold activation, partially distinguishable from physiological activation. Moreover, a glance will be given at recent proteomic approaches, which promise to improve current knowledge on blood components of transfusion interest, mainly addressing current technical hurdles (such as the analysis of membrane proteins and their reciprocal interaction).

Mechanisms coupling the hemostatic system to colitis-associated cancer

A link between inflammation and the development of cancer is well established. Indeed, approximately 15–20% of malignancies are thought to arise secondary to chronic inflammation [1]. One of the clearest illustrations of the link between inflammation and cancer is the strong association between chronic colitis (i.e., ulcerative colitis and Crohn’s disease) and colon cancer, the second leading cause of cancer deaths [2–5]. The precise mechanisms coupling inflammatory processes to colon cancer progression have not been fully defined and are likely to be numerous and complex. Chronic bowel inflammation has been proposed to drive cancer initiation by increasing intestinal epithelial cell turnover, increasing the likelihood of an epithelial carcinogenic mutation. Chronic exposure of epithelial cells to inflammatory mediators, such as reactive oxygen and nitrogen species, may also lead to mutagenesis by causing DNA damage [6,7]. Additionally, local inflammation may support the proliferation of transformed epithelial cells and the development of tumor stroma through the local release of chemokines, cytokines, growth factors, and proangiogenic factors [8]. While an association between inflammation and colon cancer progression has been well established, the specific factors that both drive local inflammatory processes and support tumor progression in this setting remain poorly defined. Interestingly, a growing body of evidence suggests that multiple hemostatic factors more traditionally associated with the maintenance of vascular integrity and prevention of blood loss are critical determinants of local inflammatory and malignant pathologies. The concept that hemostatic system components are determinants of colitis and colon cancer is supported by several clinical observations. Both colitis and colon cancer are associated with activation of

Syk and Zap-70 function redundantly to promote angioblast migration

Spleen tyrosine kinase (Syk) plays critical roles in B-cell and T-cell development, the maintenance of vascular integrity, and proper partitioning of the blood vascular and lymphatic vascular system. Here, we utilize the zebrafish as an in vivo system to demonstrate novel roles for Syk and the related kinase Zeta associated protein (Zap-70) in promoting angioblast migration. Partial knockdown of either gene results in early angiogenic delay of the intersegmental vessels, dorsal intersegmental vessel patterning defects, and partial loss of the thoracic duct. Higher dose knockdown of both genes results in little to no angiogenic sprouting of the intersegmental vessels, a phenotype which resembles knockdown of vegfa. Di-phosphorylated ERK, an effector of the vegfa pathway, is also downregulated in the aorta of syk:zap double morphants. Over-expression of syk under the control of a blood-specific or vascular-specific promoter rescues sprouting defects after loss of vegfa. Together these results suggest that syk and zap-70 function redundantly in an early progenitor to promote the migration of intersegmental vessel angioblasts and lymphangioblasts that contribute to the thoracic duct, either downstream of, or in parallel to vegfa.

Sphingosine-1-phosphate in the plasma compartment regulates basal and inflammation-induced vascular leak in mice

Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell Gi-coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models.

Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease

Endothelial progenitor cells (EPCs) play an important role in the maintenance of vascular integrity. Lipid lowering therapy (LLT) with statins may contribute to biologically relevant activities including the proliferation of endothelial cells. The physiological role of microRNA (miR)-221/222, a newly discovered class of small RNA, is closely linked to the proliferation of endothelial cells. We therefore investigated whether LLT with statins might affect miR-221/222 expression in EPCs obtained from patients with coronary artery disease (CAD). This study included 44 patients with stable CAD and 22 subjects without CAD (non-CAD). Patients with CAD were randomized to 12 months of LLT with atorvastatin (10 mg day(-1)) or pravastatin (10 mg day(-1)). EPCs were obtained from peripheral blood at baseline and after 12 months of statin therapy. Levels of miR-221/222 in EPCs were measured by real-time RT-PCR. Levels of miR-221/222 were significantly higher in the CAD group than in the non-CAD group (P < 0.01). Levels of miR-221/222 were weakly negatively correlated with EPC number in the CAD group. After 12 months of therapy, changes in lipid profiles were greater in the atorvastatin group than in the pravastatin group. LLT with atorvastatin markedly increased EPC numbers and decreased miR-221/222 levels (all P < 0.05), whereas LLT with pravastatin did not change EPC numbers or miR-221/222 levels. This study demonstrates that LLT with atorvastatin increases EPC numbers and decreases miR-221/222 levels in patients with CAD, possibly contributing to the beneficial effects of LLT with atorvastatin in this disorder.