Maintenance Of Vascular Integrity Research Articles

Atorvastatin-induced intracerebral hemorrhage is inhibited by berberine in zebrafish.

Intracerebral hemorrhage (ICH), for which there are currently no effective preventive or treatment methods, has a very high fatality rate. Statins, such as atorvastatin (ATV), are the first-line drugs for regulating blood lipids and treating hyperlipidemia-related cardiovascular diseases. However, ATV-associated ICH has been reported, although its incidence is rare. In this study, we aimed to investigate the protective action and mechanisms of berberine (BBR) against ATV-induced brain hemorrhage. We established an ICH model in zebrafish induced by ATV (2 μM) and demonstrated the effects of BBR (10, 50, and 100 μM) on ICH via protecting the vascular network using hemocyte staining and three transgenic zebrafish. BBR was found to reduce brain inflammation and locomotion injury in ICH-zebrafish. Mechanism research showed that ATV increased the levels of VE-cadherin and occludin proteins but disturbed their localization at the cell membrane by abnormal phosphorylation, which decreased the number of intercellular junctions between vascular endothelial cells (VECs), disrupting the integrity of vascular walls. BBR reversed the effects of ATV by promoting autophagic degradation of phosphorylated VE-cadherin and occludin in ATV-induced VECs examined by co-immunoprecipitation (co-IP). These findings provide crucial insights into understanding the BBR mechanisms involved in the maintenance of vascular integrity and in mitigating adverse reactions to ATV.

Abstract 4588: Vascular regulation of T-cell exclusion in melanoma

Abstract T-cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T-cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T-cell infiltration within tumor. We analyzed the spatial regulation of ANGPT2 in whole tumor cross-sections and its impact on CD8+ T-cell position, vascular integrity, and response to immunotherapy using syngeneic murine melanomas. We found that T-cell exclusion was associated with peritumoral expression of ANGPT2 and elevated vascular leakage in human and murine melanomas. Both pharmacological and genetic blockade of ANGPT2 promoted CD8+ T-cell infiltration to the tumor core, exerting antitumor effects. Importantly, the reversal of T-cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 therapy in immunogenic, therapy responsive mouse melanomas, but also rendered non-responsive tumors susceptible. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T-cell infiltration, coincided with TIE2 signaling activation and increased vascular integrity in the tumor periphery, indicating that maintenance of vascular integrity within tumors is necessary for robust response to immunotherapy. These data highlight ANGPT2/TIE2 signaling as a key mediator of T-cell exclusion and a promising target to potentiate checkpoint inhibitor efficacy in melanoma. Citation Format: Ha-Ram Park, Anahita Shiva, Portia Cummings, Richard Carvajal, Gavin Thurston, Joon-Yong An, Amanda W. Lund, Hee Won Yang, Minah Kim. Vascular regulation of T-cell exclusion in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4588.

Abstract 2599: Targeted delivery of a colchicine analogue provides synergy with ATR inhibition

Abstract Introduction: Vascular disrupting agents (VDA) are an important class of therapeutic that have yet to reach their clinical potential. Colchicine is representative of the class and is an effective disruptor of microtubule dynamics. Microtubules play an important role in the maintenance of vascular integrity, hence the ability of these agents to collapse tumour blood vessels, and consequently cause tumour necrosis. Colchicine, in addition to its vascular disrupting properties, is known to induce G2/M arrest and trigger apoptosis. ICT2588 is a non-toxic, tumour targeted prodrug of a potent colchicine analogue with reduced potential for toxicity, (cardiotoxicity in particular) due to its metabolic stability in plasma and normal tissues. The preclinical evaluation of ICT2588 to-date has been focused solely on its selective tumour vascular disrupting ability and subsequent antitumour activity. The aim of this current study was to investigate the cellular metabolism of ICT2588, examine its G2/M cell cycle arrest potential, and to explore mechanisms of synergy between ICT2588 and ATR inhibition. Method: The cellular mechanism of uptake and MT1-MMP-selective metabolism of ICT2588 was studied in MT1-MMP positive (HT1080) and negative (MCF-7) cell lines using LCMS based metabolism assays. Flow cytometry was used to analyse the effect of ICT2588 and active azademethylcolchicine (the ‘warhead’) on G2/M cell cycle arrest in both MT1-MMP positive and negative cell lines. The growth inhibitory effects of ICT2588 and its warhead either alone or in combination with ceralasertib (AZD6738, ATR inhibitor) were performed using an MTT assay in MT1-MMP positive cell lines (HT1080, MDA-MB-231, and SH-SY5Y). Synergy Finder (https://synergyfinder.fimm.fi) was used to evaluate combinations. Results: The expression of MT1-MMP correlates with selective metabolism of ICT2588 and subsequent selective release of its warhead in HT1080 cells, but not MCF-7 cells. This metabolism is inhibited by an MT1-MMP inhibitor. Metabolism of ICT2588 induced G2/M arrest and triggered apoptosis in HT1080 cells, but not in MCF-7 cells, though the warhead induced G2/M arrest and triggered apoptosis in both cell lines. ICT2588 was synergistic with AZD6738 in all MT1-MMP positive cell lines, contrary to the antagonism observed with the warhead and AZD6738. These findings will be explored. Conclusion: ICT2588 is metabolised by only MT1-MMP expressing cells, releasing the potent colchicine analogue in a targeted-manner, which synergised with ATR inhibition in a way not observed when the warhead was administered alone. This in vitro data has important potential implications for possible combinations of ICT2588 and DNA damage repair inhibitors in the clinic. Citation Format: Francis M. Barnieh, Goreti Ribeiro Morais, Paul M. Loadman, Robert A. Falconer. Targeted delivery of a colchicine analogue provides synergy with ATR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2599.

Single-cell mRNA analysis of colon tissue during DSS colitis in CX3CR1cre/+EP4fl/fl mice suggests PGE2 signaling via EP4 promotes survival of protective macrophages

Abstract Prostaglandin E2 signaling through its receptor EP4 on macrophages (MPs) protects against DSS-induced colitis, however the mechanisms involved are not clear. Here we perform single cell RNA sequencing on the colons of CX3CR1cre/+EP4fl/fl(Cre+) mice and CX3CR1+/+EP4fl/fl(Cre−) littermate controls at days 0, 3 and 7 following DSS treatment. We identified monocyte (MO), MP, DC1, DC2 and pre-DC populations, with MPs clustering into Hes1hi, Mrc1hi Siglec1(CD169)hi, Retnlahi, and Il1r2hiCd80hi populations. With colitis there was a dramatic increase in the proportions of inflammatory MOs and a loss of all mature MPs, along with an increase in cDC2 but not cDC1 in both mouse strains. However, in Cre+ mice, which develop worse disease with universal GI bleeding, the loss of Mrc1hi Siglec1hi MPs was accelerated together with an increased proportion of Il1r2hiCd80hi MPs compared to Cre− littermates. Analysis of DEGs showed the Il1r2hiCd80hi MP cluster expresses high levels of leukocyte chemoattractants, suggesting that PGE2 may prevent leukocyte recruitment during inflammation. Cre+ mice also had fewer Retnlahi macrophages than Cre− mice both at days 0 and 7 post DSS. Genes highly expressed in the Retnlahi cluster are associated with wound healing and maintenance of vascular integrity indicating their loss could account for the enhanced GI bleeding in the inflamed Cre+ mice. This is the first detailed scmRNA analysis of immune cell populations involved in DSS colitis. While additional investigation is necessary, the data suggest a role for PGE2 signaling via EP4 on MPs in preventing excessive inflammatory cell infiltration, decreasing vascular permeability, and contributing to tissue repair by maintaining protective MP populations.

Heparan sulfate mimetic fucoidan restores the endothelial glycocalyx and protects against dysfunction induced by serum of COVID-19 patients in the intensive care unit

Accumulating evidence proves that endothelial dysfunction is involved in coronavirus disease 2019 (COVID-19) progression. We previously demonstrated that the endothelial surface glycocalyx has a critical role in maintenance of vascular integrity. Here, we hypothesised that serum factors of severe COVID-19 patients affect the glycocalyx and result in endothelial dysfunction.We included blood samples of 32 COVID-19 hospitalised patients at the Leiden University Medical Center, of which 26 were hospitalised in an intensive care unit (ICU) and six on a non-ICU hospital floor; 18 of the samples were obtained from convalescent patients 6 weeks after hospital discharge, and 12 from age-matched healthy donors (control) during the first period of the outbreak. First, we determined endothelial (angiopoietin 2 (ANG2)) and glycocalyx degradation (soluble thrombomodulin (sTM) and syndecan-1 (sSDC1)) markers in plasma.In the plasma of COVID-19 patients, circulating ANG2 and sTM were elevated in patients in the ICU. Primary lung microvascular endothelial cell (HPMEC) and human glomerular microvascular endothelial cell (GEnC) cultured in the presence of these sera led to endothelial cell glycocalyx degradation, barrier disruption, inflammation and increased coagulation on the endothelial surface, significantly different compared to healthy control and non-ICU patient sera. These changes could all be restored in the presence of fucoidan.In conclusion, our data highlight the link between endothelial glycocalyx degradation, barrier failure and induction of a procoagulant surface in COVID-19 patients in ICU which could be targeted earlier in disease by the presence of heparan sulfate mimetics.

Human Endothelial Progenitor Cells Protect the Kidney against Ischemia-Reperfusion Injury via the NLRP3 Inflammasome in Mice.

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and progression to chronic kidney disease (CKD). However, no effective therapeutic intervention has been established for ischemic AKI. Endothelial progenitor cells (EPCs) have major roles in the maintenance of vascular integrity and the repair of endothelial damage; they also serve as therapeutic agents in various kidney diseases. Thus, we examined whether EPCs have a renoprotective effect in an IRI mouse model. Mice were assigned to sham, EPC, IRI-only, and EPC-treated IRI groups. EPCs originating from human peripheral blood were cultured. The EPCs were administered 5 min before reperfusion, and all mice were killed 72 h after IRI. Blood urea nitrogen, serum creatinine, and tissue injury were significantly increased in IRI mice; EPCs significantly improved the manifestations of IRI. Apoptotic cell death and oxidative stress were significantly reduced in EPC-treated IRI mice. Administration of EPCs decreased the expression levels of NLRP3, cleaved caspase-1, p-NF-κB, and p-p38. Furthermore, the expression levels of F4/80, ICAM-1, RORγt, and IL-17RA were significantly reduced in EPC-treated IRI mice. Finally, the levels of EMT-associated factors (TGF-β, α-SMA, Snail, and Twist) were significantly reduced in EPC-treated IRI mice. This study shows that inflammasome-mediated inflammation accompanied by immune modulation and fibrosis is a potential target of EPCs as a treatment for IRI-induced AKI and the prevention of progression to CKD.

CDH5, a Possible New Candidate Gene for Genetic Testing of Lymphedema.

Background: Expressed by endothelial cells, CDH5 is a cadherin involved in vascular morphogenesis and in the maintenance of vascular integrity and lymphatic function. The main purpose of our study was to identify distinct variants of the CDH5 gene that could be associated with lymphatic malformations and predisposition for lymphedema. Methods and Results: We performed Next Generation Sequencing of the CDH5 gene in 235 Italian patients diagnosed with lymphedema but who tested negative for variants in known lymphedema genes. We detected six different variants in CDH5 five missense and one nonsense. We also tested available family members of the probands. For family members who carried the same variant as the proband, we performed lymphoscintigraphy to detect any lymphatic system abnormalities. Variants were modeled in silico. The results showed that CDH5 variants may contribute to the onset of lymphedema, although further in vitro studies are needed to confirm this hypothesis. Conclusions: Based on our findings, we propose CDH5 as a new gene that could be screened in patients with lymphedema to gather additional evidence.

Abstract P408: Mesenchymal Stem Cell-derived Angptl4 Improves Endothelial Permeability And Resolution Of Inflammation In Atherosclerosis

Objective: Given the fundamental contribution of inflammation to atherosclerosis, we studied the effect of ANGPTL4 in regulating vascular lesions during atherosclerosis. Methods and Results: We analyzed plasma levels of ANGPTL4 and found that acute myocardial infarction (AMI) patients with higher levels of ANGPTL4 had fewer vascular events than did patients with lower ANGPTL4 levels ( p <0.05). Moreover, in AMI patients with heart failure (HF) at admission, the recurrence of HF was lower in patients with a higher level of ANGPTL4. We then investigated the therapeutic application of ANGPTL4 in an atherosclerosis model. Apoe-/- mice fed a high-fat diet were injected with PBS or ANGPTL4 protein (2 μg per mouse, i.p. ) three times per week for 7 weeks. En face staining and mRNA analysis showed that plaque size, necrotic core area, lipid accumulation, and inflammatory molecules were greatly reduced in the ANGPTL4 group. Aortic permeability, measured by leakage of Evans blue dye, was significantly decreased in the ANGPTL4 group. The induction of pro-inflammatory mediators was significantly inhibited in endothelial cells, vascular smooth muscle cells, and macrophages by ANGPTL4 treatment. Endothelial Krüppel-like factor 2 (KLF2) and VE-cadherin were restored to contribute to maintenance of vascular integrity by ANGPTL4 treatment. Elevated levels of circulating leptin, interleukin-6, and interleukin-1β were profoundly reduced. Most importantly, the fibrous cap was significant thicker in the ANGPTL4 group than in the PBS group. Conclusions: ANGPTL4 treatment attenuated atherogenesis, which suggests that targeting vascular stability and inflammation may serve as a novel therapeutic strategy to prevent and treat atherosclerosis. More importantly, ANGPTL4 treatment inhibits necrotic core formation in lesions, leading to the formation of more stable plaques as evidenced by increased fibrous cap thickness.

Platelet Innate Immune Receptors and TLRs: A Double-Edged Sword.

Platelets are hematopoietic cells whose main function has for a long time been considered to be the maintenance of vascular integrity. They have an essential role in the hemostatic response, but they also have functional capabilities that go far beyond it. This review will provide an overview of platelet functions. Indeed, stress signals may induce platelet apoptosis through proapoptotis or hemostasis receptors, necrosis, and even autophagy. Platelets also interact with immune cells and modulate immune responses in terms of activation, maturation, recruitment and cytokine secretion. This review will also show that platelets, thanks to their wide range of innate immune receptors, and in particular toll-like receptors, and can be considered sentinels actively participating in the immuno-surveillance of the body. We will discuss the diversity of platelet responses following the engagement of these receptors as well as the signaling pathways involved. Finally, we will show that while platelets contribute significantly, via their TLRs, to immune response and inflammation, these receptors also participate in the pathophysiological processes associated with various pathogens and diseases, including cancer and atherosclerosis.

Noval Insights into the Role of Platelet Angiogenic Growth Factors on the Regulation of Normal Vascular Development

The role of platelets as angiogenic regulators has been the subject of intense research over the last decade, and has led to the recognition of platelets as the predominant source of key angiogenic growth factors (AGFs) in the circulation. Through the regulated delivery of these factors, platelets contribute to wound healing, maintenance of vascular integrity, and tumor angiogenesis. Prior studies have shown that thrombocytopenia in adult mice leads to hemorrhages primarily from structurally abnormal new blood vessels, both in in vivo angiogenesis assays and inside tumors. The rapid growth during fetal and neonatal life is associated with a developmental stage-specific need for accelerated angiogenesis and vascular development. However, whether and how platelets participate in normal fetal/neonatal vascular development is unknown, except for their role in blood/lymphatic separation. We hypothesized that platelets would be important for normal fetal/neonatal vascular development, and that their AGF profile would be different from that of adult platelets. As a first step, we quantified five key AGFs (VEGF-A, bFGF, ANG-1, PDGF-BB and TGF-b) by ELISA in platelet lysates and platelet-poor plasma from term cord blood samples (CB; n=12) and from the peripheral blood of healthy adult volunteers (PB; n=8). These studies demonstrated that CB platelets, just like PB platelets, are the major source of AGFs in the circulation, carrying more than 80% of the levels of these AGFs present in the blood. However, the angiogenic profiles were different in platelets of different developmental origins. Fetal/neonatal platelets contained ~70% more bFGF and VEGF-A than adult platelets (p<0.01 for both), but had significantly lower levels of ANG-1, PDGF-BB and TGF-β1 (p<0.05 vs. adult platelets). VEGF-A and bFGF are essential for the proliferation and differentiation of endothelial cells, while ANG-1, PDGFs and TGF-b concert mural cell coverage, basement membrane formation and vascular maturation, thus stabilizing and “strengthening” the vasculature. Based on these characteristics, we hypothesized that neonatal and adult platelets would have different biological effects. To test this, we first assessed the effects of CB- vs. PB-derived platelet lysates on human umbilical vascular endothelial cells (HUVEC), in a tube formation assay. In these experiments, CB platelets more potently stimulated angiogenesis, quantified as number of capillary branch points at 4, 5, and 6 hours, than PB platelets. Subsequently, we compared the proliferative effects of CB vs. PB platelet lysates on human primary CB-derived endothelial progenitor cells (EPCs) and amniotic membrane-derived mesenchymal stromal cells (MSCs). Consistent with the effects on HUVEC cells, CB platelet lysates induced approximately 50% more proliferation of EPCs, compared to PB platelet lysates (p<0.001). In contrast, PB platelet lysates were approximately twice more potent in promoting MSCs growth (p<0.001 vs. CB platelets). Since EPCs give rise to the vascular endothelium and MSCs differentiate into mural cells, these results strongly suggested that the AGF content in fetal/neonatal platelets promotes rapid angiogenesis at the expense of a stable vasculature, matching the developmental needs of rapidly growing fetuses and neonates. Finally, since birth is associated with an abrupt change in oxygen concentration (from the profoundly hypoxic intrauterine to the normoxic extrauterine environment), we assessed whether oxygen concentration would modulate the VEGF content in CB-derived cultured MKs (and thus platelets). Consistent with this hypothesis, CB MKs cultured in 5% oxygen released significantly more VEGF in the medium compared to MKs cultured in 20% oxygen (p<0.05). Our findings strongly suggest that the platelet AGF profile changes from neonatal to adult life to help regulate vascular development at different stages of life, and suggest that it might also change in response to environmental factors such as oxygen concentration. Whether a change in platelet angiogenic profile associated with the sudden increase in oxygen concentration during preterm birth contributes to diseases of prematurity characterized by disrupted vascular development (i.e. retinopathy of prematurity) requires further investigation. DisclosuresSola-Visner:Sysmex Inc.: Research Funding.

MO334ERYTHROPOIETIN-STIMULATED HUMAN ENDOTHELIAL PROGENITOR CELLS AMELIORATE INFLAMMATION AND FIBROSIS VIA INFLAMMASOME IN ISCHEMIA/REPERFUSION-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Ischemia/reperfusion-induced AKI (IR-AKI) is a major cause of AKI and progress to chronic kidney disease. But an effective therapeutic intervention for IR-AKI is not established yet. Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is significantly upregulated during hypoxia. Endothelial progenitor cells (EPCs) are derived from the bone marrow or tissue-resident cells and play major roles in the maintenance of vascular integrity and the repair of endothelial damage. So, we investigated if EPO-stimulated human EPCs could have the renoprotective effects in an IR-AKI mouse model. Method EPCs originated from human peripheral blood were cultured with EPO (10 IU/mL). Mice were assigned to sham, IR only groups, IR with EPC, and IR with EPO-treated EPC. EPCs (5x105 cells, tail vein) were administered twice at 30 min prior to bilateral renal artery occlusion, and 5 min before reperfusion, with all mice sacrificed 24 h after IR-AKI. Results Both EPCs and EPO-treated EPCs significantly attenuated the renal dysfunction associated with IR-AKI, as well as tissue injury. Apoptotic cell death and oxidative stress were significantly reduced in EPC and EPO-treated EPC mice. Expression of PCNA, ICAM-1, MCP-1 and α-SMA were also significantly reduced in EPC and EPO-treated EPC mice. Furthermore, the expression of NLRP3 and caspase-1 via the activation of NF-κB signaling pathways were significantly reduced in EPC and EPO-treated EPC mice. These results show more effective in EPO-treated EPC than EPC alone and suggest EPO might be involved in the development of EPC. Conclusion This study provides that inflammasome-mediated inflammation and fibrosis might be a potential target of EPC as a treatment for IR-AKI.

Platelets, Thrombocytosis, and Ovarian Cancer Prognosis: Surveying the Landscape of the Literature.

Platelets are critical components of a number of physiologic processes, including tissue remodeling after injury, wound healing, and maintenance of vascular integrity. Increasing evidence suggests that platelets may also play important roles in cancer. In ovarian cancer, thrombocytosis, both at the time of initial diagnosis and at recurrence, has been associated with poorer prognosis. This review describes current evidence for associations between thrombocytosis and ovarian cancer prognosis and discusses the clinical relevance of platelet count thresholds and timing of assessment. In addition, we discuss several mechanisms from in vitro, in vivo, and clinical studies that may underlie these associations and recommend potential approaches for novel therapeutic targets for this lethal disease.

Influence of different methods and anticoagulants on platelet parameter measurement

Abstract Platelets are the smallest and perhaps the most versatile components of human blood. Besides their role in coagulation and the maintenance of vascular integrity, they are involved in many physiological processes, ranging from immune response and leukocyte recruitment to the production of antimicrobial peptides and immune-suppressive factors like TGF-β. These versatile abilities make platelets interesting for researchers from different disciplines. However, beside profound investigation into platelets’ physiological role, there is a need for correct, standardized and thus reproducible quantification of platelet parameters. Mean platelet volume (MPV) is a widespread prognostic marker for several conditions, such as, acute coronary syndrome, chronic kidney disease and liver cirrhosis. Platelet activation is regarded as a marker for inflammatory processes, for example in autoimmune diseases such as type-1 diabetes, systemic lupus erythematosus and rheumatoid arthritis. The monitoring of platelet function is relevant for patients receiving antiplatelet medication. Platelet parameter measurement is affected by the choice of in vitro anticoagulant, the measurement technology and the time delay after sampling. This review focuses on the pre-analytical variability that arises as a result of the use of different in vitro anticoagulants and analyzer technologies when determining platelet parameters, since, even approximately 180 years after the discovery of platelets, there is still no standardized procedure.

Relationship between Microfibrillar-Associated Protein 4 Levels and Subclinical Myocardial Damage in Chronic Kidney Disease

Introduction: Chronic kidney disease (CKD) is a widespread health problem, in which mortality is most frequently due to cardiovascular diseases. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein. MFAP4 is involved in several biological processes, particularly the maintenance of vascular integrity and extracellular matrix remodeling. Our review of the literature revealed no data concerning MFAP4 levels in CKD and its relationship with myocardial functions. Objective:The purpose of this study was therefore to investigate MFAP4 levels in CKD, parameters affecting these, and the relationship with myocardial functions. Materials and Methods: Seventy-nine CKD patients and 30 healthy controls were included in the study. Routine biochemical tests and echocardiography were performed once demographic data had been recorded. Blood specimens were collected for MFAP4 analysis, and the results were subjected to statistical analysis. Results: MFAP4 levels were significantly higher in the patient group than in the control group (p< 0.001). Doppler parameters revealed more frequent LV diastolic impairment in the patient group. Tissue Doppler systolic velocity and global longitudinal strain were significantly impaired, revealing the subclinical LV systolic dysfunction in CKD patients. MFAP4 elevation in the patient group was positively correlated with aortic root (AR), global circumferential strain (GCS), and GCS rate. Conclusion: Our results showed MFAP4 elevation in CKD for the first time in the literature, and that this elevation may be related to GCS and AR dilation. We think that, once supported by further studies, MFAP4 may constitute a marker in the evaluation of myocardial functions in CKD.

C-Src controls stability of sprouting blood vessels in the developing retina independently of cell-cell adhesion through focal adhesion assembly.

ABSTRACTEndothelial cell adhesion is implicated in blood vessel sprout formation, yet how adhesion controls angiogenesis, and whether it occurs via rapid remodeling of adherens junctions or focal adhesion assembly, or both, remains poorly understood. Furthermore, how endothelial cell adhesion is controlled in particular tissues and under different conditions remains unexplored. Here, we have identified an unexpected role for spatiotemporal c-Src activity in sprouting angiogenesis in the retina, which is in contrast to the dominant focus on the role of c-Src in the maintenance of vascular integrity. Thus, mice specifically deficient in endothelial c-Src displayed significantly reduced blood vessel sprouting and loss in actin-rich filopodial protrusions at the vascular front of the developing retina. In contrast to what has been observed during vascular leakage, endothelial cell-cell adhesion was unaffected by loss of c-Src. Instead, decreased angiogenic sprouting was due to loss of focal adhesion assembly and cell-matrix adhesion, resulting in loss of sprout stability. These results demonstrate that c-Src signaling at specified endothelial cell membrane compartments (adherens junctions or focal adhesions) control vascular processes in a tissue- and context-dependent manner.

Platelet lamellipodium formation is not required for thrombus formation and stability

AbstractDuring platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.

Mast Cell Degranulation Decreases Lipopolysaccharide-Induced Aortic Gene Expression and Systemic Levels of Interleukin-6 In Vivo.

Mast cells play an important role in immunomodulation and in the maintenance of vascular integrity. Interleukin-6 (IL-6) is one of the key biomarkers and therapeutic target in systemic vasculitis. The objective of the current study is to describe the role of mast cells in arterial IL-6 homeostasis. Eight- to ten-week-old male C57BL/6 (wild-type) mice were injected with either (a) saline, (b) compound 48/80 (a systemic mast cell degranulating agent), (c) lipopolysaccharide (LPS), or (d) a combination of C48/80 and LPS. Twenty-four hours after the injections, mice were sacrificed and serum samples and aortic tissues were analyzed for determining inflammatory response and cytokine expression profile. The results revealed that induction of mast cell degranulation significantly lowers serum IL-6 levels and aortic expression of IL-6 in LPS-treated mice. Significantly higher aortic expression of toll-like receptor-2 (TLR-2) and TNF-α was seen in the LPS and LPS+C48/80 groups of mice compared to controls. Aortic expression of TLR-4 was significantly decreased in LPS+C48/80 compared to C48/80 alone. LPS+C48/80-treated mice presented with a 3-fold higher aortic expression of suppressor of cytokine signaling (SOCS-1) compared to saline-injected groups. The inhibition of LPS-induced increase in serum IL-6 levels by mast cell degranulation was not seen in H1R knockout mice which suggests that mast cell-derived histamine acting through H1R may participate in the regulatory process. To examine whether the mast cell-mediated downregulation of LPS-induced IL-6 production is transient or cumulative in nature, wild-type mice were injected serially over a period of 10 days (5 injections) and serum cytokine levels were quantified. We found no significant differences in serum IL-6 levels between any of the groups. While mice injected with C48/80 or LPS had higher IL-10 compared to vehicle-injected mice, there was no difference between C48/80- and LPS+C48/80-injected mice. In conclusion, in an in vivo setting, mast cells appear to partially and transiently regulate systemic IL-6 homeostasis. This effect may be regulated through increased systemic IL-10 and/or aortic overexpression of SOCS-1.

Regulation of PLPP3 gene expression by NF-κB family transcription factors

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.

Sphingosine 1-phosphate and inflammation.

AbstractSphingosine 1-phosphate (S1P), a sphingolipid mediator, regulates various cellular functions via high-affinity G protein-coupled receptors, S1P1-5. The S1P-S1P receptor signaling system plays important roles in lymphocyte trafficking and maintenance of vascular integrity, thus contributing to the regulation of complex inflammatory processes. S1P is enriched in blood and lymph while maintained low in intracellular or interstitial fluids, creating a steep S1P gradient that is utilized to facilitate efficient egress of lymphocytes from lymphoid organs. Blockage of the S1P-S1P receptor signaling system results in a marked decrease in circulating lymphocytes because of a failure of lymphocyte egress from lymphoid organs. This provides a basis of immunomodulatory drugs targeting S1P1 receptor such as FTY720, an immunosuppressive drug approved in 2010 as the first oral treatment for relapsing-remitting multiple sclerosis. The S1P-S1P receptor signaling system also plays important roles in maintenance of vascular integrity since it suppresses sprouting angiogenesis and regulates vascular permeability. Dysfunction of the S1P-S1P receptor signaling system results in various vascular defects, such as exaggerated angiogenesis in developing retina and augmented inflammation due to increased permeability. Endothelial-specific deletion of S1P1 receptor in mice fed high-fat diet leads to increased formation of atherosclerotic lesions. This review highlights the importance of the S1P-S1P receptor signaling system in inflammatory processes. We also describe our recent findings regarding a specific S1P chaperone, apolipoprotein M, that anchors to high-density lipoprotein and contributes to shaping the endothelial-protective and anti-inflammatory properties of high-density lipoprotein.

Platelet Btk is Required for Maintaining Lung Vascular Integrity during Murine Pneumococcal Pneumosepsis.

Platelet Bruton's tyrosine kinase (Btk) is an essential signalling protein for the collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type-lectin-like receptor-2, which are platelet receptors implicated in the maintenance of vascular integrity during inflammation. Moreover, platelets, platelet GPVI and Btk are important for host defence during murine bacterial pneumosepsis. The aim of this study was to determine the role of platelet Btk in vascular integrity and host defence during murine pneumosepsis caused by the common human pathogens Streptococcus pneumoniae and Klebsiella pneumoniae. Using the Cre-loxP system, male platelet-specific Btk-deficient mice (PF4creBtkfl/Y) were created. Similar to platelets from total Btk-deficient mice, platelets from PF4creBtkfl/Y mice showed abrogated aggregation and P-selectin expression when stimulated with the GPVI ligand cross-linked collagen-related peptide. Upon infection with S. pneumoniae, PF4creBtkfl/Y mice showed increased lung bleeding, but unimpaired anti-bacterial defence. During pneumosepsis evoked by K. pneumoniae, platelet Btk deficiency was not associated with lung bleeding and did not impact on host defence, even when platelet function was further compromised by blocking secondary platelet activation by the P2Y12 receptor antagonist clopidogrel. Together, these data indicate that, while platelet Btk is not important for anti-bacterial defence in pneumosepsis, its role in maintaining vascular integrity in the lung depends on the causative pathogen.