Apoptotic Endothelial Cells Research Articles

A prostacyclin analog prevents the regression of renal microvascular network by inhibiting mitochondria-dependent apoptosis in the kidney of rat progressive glomerulonephritis

We have previously demonstrated that renoprotective effects of a prostacyclin analog, beraprost sodium, on the kidney of anti-glomerular basement membrane glomerulonephritis (GN) rats. The aim of this study is to address the renoprotection mechanism of beraprost sodium, especially in the terminal stage of GN. Beraprost sodium was orally administrated from 2 to 7 weeks after induction of GN, and renal function, morphology, protein and mRNA levels were analyzed. We found the beraprost sodium treatment suppressed the structural regression of renal microvascular network and decline of renal blood flow occurred in the kidney of GN rats. To address the mechanism of the structural maintenance, we focused on apoptosis because the increased number of apoptotic renal microvascular endothelial cells and tubular epithelial cells was observed in the kidneys of GN rats as compared with normal and beraprost sodium treated rats. Protein and mRNA analyses demonstrated that mitochondria-dependent apoptotic pathway was activated in the kidneys of GN rats, and beraprost sodium suppressed the activation by modulating the expression patterns of pro- and anti-apoptotic factors. These results suggest that inhibition of mitochondria-dependent apoptosis of renal cells in GN kidney and consequent maintenance of renal functional structures, including microvascular network might contribute to the renoprotective effect of beraprost sodium in GN.

Circulating endothelial microparticles involved in lung function decline in a rat exposed in cigarette smoke maybe from apoptotic pulmonary capillary endothelial cells.

Plasma levels of endothelial microparticles (EMPs), small membrane vesicles, shed from activated or apoptotic endothelial cells are elevated in patients with COPD and in smokers with normal lung function. Whether plasma EMPs levels are elevated in a rat exposed in cigarette smoke, whether the elevated EMPs derived from pulmonary endothelial cell apoptosis, and the relationship between EMP and lung function are obscure. All 60 wister rats were divided into six groups, three groups of ten rats were exposed to cigarette smoke of ten non-filter cigarettes per day, 5 days a week, using a standard smoking machine (Beijing BeiLanBo Company, China) for a period of 2, 4 and 6 months (n=10, respectively). Age-matched three control groups were sham-smoked. Pulmonary function parameters, including the ratio of forced expiratory volume in 0.3 second over forced vital capacity (FEV0.3/FVC) and dynamic compliance (Cdyn), were tested at the end of each period (2, 4, 6 months). Blood samples were collected and platelet-free plasma was isolated. Then CD42b-/CD31+ EMPs were analysed by flow cytometry. In parallel, lungs were removed and Colocalization with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), Hoeschts and CD31 was performed to evaluate pulmonary capillaries-specific apoptosis and identify the origins of the EMPs. At 2, 4 and 6 months, in comparison with control groups, rats in cigarette smoke exposed groups had a significant increase in CD42b-/CD31+ EMPs (P<0.001, P<0.001, P<0.001, respectively), and Pulmonary function indicated that FEV0.3/FVC (P<0.05, P<0.01, P<0.01, respectively) and Cdyn (P<0.01, P<0.001, P<0.001 respectively) decreased. At the same time, CD42b-/CD31+ EMP counts were negatively correlated with Cdyn (P<0.05). Moreover, in vivo, TUNEL-positive cells co-localized with CD31 in whole lung tissue demonstrated a sequence of apoptosis signal in the cigarette smoke exposed groups. CD42b-/CD31+ EMPs may be a potential biomarker for indicating the severity of impairment of pulmonary function in the rats exposed cigarette smoke. The increased EMPs may derive from pulmonary capillaries-specific apoptosis.

CLM29, a multi-target pyrazolopyrimidine derivative, has anti-neoplastic activity in medullary thyroid cancer in vitro and in vivo

CLM29 (a pyrazolo[3,4-d]pyrimidine, that inhibits RET, epidermal growth factor receptor, vascular endothelial growth factor receptor, and has an anti-angiogenic activity) has anti-neoplastic activity in papillary dedifferentiated thyroid cancer. Here we tested CLM29 in medullary thyroid cancer (MTC), in primary MTC cells (P-MTC) obtained at surgery, and in TT cells harboring (C634W) RET mutation.CLM29 (10, 30, 50μM) inhibited significantly (P<0.001) the proliferation, and increased the percentage of apoptotic P-MTC, TT and human dermal microvascular endothelial cells. The inhibition of proliferation by CLM29 was similar in P-MTC cells with/without RET mutation. TT cells were injected sc in CD nu/nu mice, and tumor masses became detectable between 20 and 30days after xenotransplantation; CLM29 (50mg/kg/die) reduced significantly tumor growth and weight, and microvessel density. The anti-tumor activity of CLM29 has been shown in MTC in vitro, and in vivo, opening the way to a future clinical evaluation.

Effects of high intensity training and high volume training on endothelial microparticles and angiogenic growth factors.

AimsEndothelial microparticles (EMP) are complex vesicular structures shed from activated or apoptotic endothelial cells. As endurance exercise affects the endothelium, the objective of the study was to examine levels of EMP and angiogenic growth factors following different endurance exercise protocols.Methods12 subjects performed 3 different endurance exercise protocols: 1. High volume training (HVT; 130 min at 55% peak power output (PPO); 2. 4×4 min at 95% PPO; 3. 4×30 sec all-out. EMPs were quantified using flow cytometry after staining platelet-poor-plasma. Events positive for Annexin-V and CD31, and negative for CD42b, were classified as EMPs. Vascular endothelial growth factor (VEGF), migratory inhibiting factor (MIF) and hepatocyte growth factor (HGF) were determined by ELISA technique. For all these measurements venous blood samples were taken pre, 0′, 30′, 60′ and 180′ after each intervention. Furthermore, in vitro experiments were performed to explore the effect of collected sera on target endothelial functions and MP uptake capacities.ResultsVEGF and HGF significantly increased after HIT interventions. All three interventions caused a significant decrease in EMP levels post exercise compared to pre values. The sera taken after exercise increased the uptake of EMP in target endothelial cells compared to sera taken under resting conditions, which was shown to be phosphatidylserin-dependent. Increased EMP uptake was associated with an improved protection of target cells against apoptosis. Sera taken prior and after exercise promoted target endothelial cell migration, which was abrogated after inhibition of VEGF.ConclusionPhysical exercise leads to decreased EMP levels and promotes a phosphatidylserin-dependent uptake of EMP into target endothelial cells, which is associated with a protection of target cells against apoptosis.

Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria.

We hypothesized that the glycocalyx, which is important for endothelial integrity, is lost in severe malaria. C57BL/6 mice were infected with Plasmodium berghei ANKA, resulting in cerebral malaria, or P. chabaudi AS, resulting in uncomplicated malaria. We visualized the glycocalyx with transmission electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested that glycocalyx disruption preceded cerebral manifestations. The contribution of this loss to pathogenesis should be studied further.

Human mesenchymal stem cells selectivelly recognize and adhere to apoptotic endothelial cells

Human mesenchymal stem cells selectivelly recognize and adhere to apoptotic endothelial cells

Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Methamphetamine (METH) is a psychostimulant with high abuse potential and severe neurotoxicity. Recent studies in animal models have indicated that METH can impair the blood–brain barrier (BBB), suggesting that some of the neurotoxic effects resulting from METH abuse could be due to barrier disruption. We report here that while chronic exposure to METH disrupts barrier function of primary human brain microvascular endothelial cells (HBMECs) and human umbilical vein endothelial cells (HUVECs), an early pro-survival response is observed following acute exposure by induction of autophagic mechanisms. Acute METH exposure induces an early increase in Beclin1 and LC3 recruitment. This is mediated through inactivation of the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70S6K pathway, and upregulation of the ERK1/2. Blockade of Kappa opioid receptor (KOR), and treatment with autophagic inhibitors accelerated METH-induced apoptosis, suggesting that the early autophagic response is a survival mechanism for endothelial cells and is mediated through the kappa opioid receptor. Our studies indicate that kappa opioid receptor can be therapeutically exploited for attenuating METH-induced BBB dysfunction.

Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia–reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer

Mesenchymal stem cells can be used as a novel treatment of ischemic vascular disease; however, their therapeutic effect and mechanism of action require further evaluation. Mitochondrial dysfunction has core functions in ischemia–reperfusion injury of the microvascular network. A recent discovery has shown that intercellular communication using tunneling nanotubes can transfer mitochondria between adjacent cells. This study aimed to investigate the tunneling nanotube mechanisms that might be involved in stem cell-mediated mitochondrial rescue of injured vascular endothelial cells. Using laser scanning confocal microscopy, mitochondrial transfer via a tunneling nanotube-like structure was detected between mesenchymal stem cells and human umbilical vein endothelial cells. Oxygen glucose deprivation and reoxygenation were performed on human umbilical vein endothelial cells, which induced mitochondrial transfer through tunneling nanotube-like structures to become frequent and almost unidirectional from mesenchymal stem cells to injured endothelial cells, thereby resulting in the rescue of aerobic respiration and protection of endothelial cells from apoptosis. We found that the formation of tunneling nanotube-like structures might represent a defense and rescue mechanism through phosphatidylserines exposed on the surface of apoptotic endothelial cells and stem cell recognition. Our data provided evidence that stem cells can rescue damaged vascular endothelial cells through a mechanism that has not yet been identified.

Impaired Pattern of Endothelial Derived Microparticles in Heart Failure Patients

Chronic heart failure (CHF) remains a leading cause of cardiovascular morbidity and mortality worldwide that is characterized pandemic features. Metabolic diseases, neurohumoral state, and systemic inflammatory response are considered the origin of microvascular endothelial cell inflammation that leads to development of CHF and supporting cardiac remodelling and vascular dysfunction. Endothelial-derived microparticles (EMPs) are considered a novel biological marker of endothelial injury and vascular tone disorders. Biological effect of EMPs realizes through supporting of cell-to-cell cross-talking mediated by transport microRNA, active molecules, hormones, peptides, regulator proteins, etc. EMPs derived from activated endothelial cells may play a pivotal role in the vascular remodelling and endothelial reparation. In opposite, EMPs originated from apoptotic endothelial cells are considered a direct trigger of vascular injury. The review is addressed to discussion of one of possible mechanism of effect realizing of endothelial-derived microparticles different origin in heart failure affected endogenous reparation and tissue injury.

Apoptosis in capillary endothelial cells in ageing skeletal muscle

The age-related loss of skeletal muscle mass and function (sarcopenia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonuclei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostaining for dystrophin, paired box protein-7 (Pax7) or laminin-2α, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age-related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre-associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7+ satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age-dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.

Caspases and p38 MAPK Regulate Endothelial Cell Adhesiveness for Mesenchymal Stem Cells

Mesenchymal stem cells natively circulating or delivered into the blood stream home to sites of injury. The mechanism of mesenchymal stem cell homing to sites of injury is poorly understood. We have shown that the development of apoptosis in endothelial cells stimulates endothelial cell adhesiveness for mesenchymal stem cells. Adhesion of mesenchymal stem cells to apoptotic endothelial cells depends on the activation of endothelial caspases and p38 MAPK. Activation of p38 MAPK in endothelial cells has a primary effect while the activation of caspases potentiates the mesenchymal stem cell adhesion. Overall, our study of the mesenchymal stem cell interaction with endothelial cells indicates that mesenchymal stem cells recognize and specifically adhere to distressed/apoptotic endothelial cells.

Endothelial Microparticle–Mediated Transfer of MicroRNA-126 Promotes Vascular Endothelial Cell Repair via SPRED1 and Is Abrogated in Glucose-Damaged Endothelial Microparticles

Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Three‐dimensional morphometric comparison of normal and apoptotic endothelial cells based on laser scanning confocal microscopy observation

Three-dimensional (3D) morphometric analysis of cellular and subcellular structures provides an effective method for spatial cell biology. Here, 3D cellular and nuclear morphologies are reconstructed to quantify and compare morphometric differences between normal and apoptotic endothelial cells. Human umbilical vein endothelial cells (HUVECs) are treated with 60 μM H2 O2 to get apoptotic cell model and then a series of sectional images are acquired from laser scanning confocal microscopy. The 3D cell model containing plasma membrane and cell nucleus is reconstructed and fused utilizing three sequential softwares or packages (Mimics, Geomagic, and VTK). The results reveal that H2 O2 can induce apoptosis effectively by regulating the activity of apoptosis-related biomolecules, including pro-apoptotic factors p53 and Bax, and anti-apoptotic factor Bcl-2. Compared with the normal HUVECs, the apoptotic cells exhibit significant 3D morphometric parameters (height, volume and nucleus-to-cytoplasm ratio) variation. The present research provides a new perspective on comparative quantitative analysis associated with cell apoptosis and points to the value of LSCM as an objective tool for 3D cell reconstruction.

Endothelial microparticle-mediated transfer of microRNA126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles

Introduction: Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. Here, we sought to investigate whether endothelial microparticles (EMP), released from apoptotic endothelial cells (ECs), influence EC repair. Methods and results: EMP were generated from human coronary endothelial cells (HCAEC) and characterized by confocal microscopy and flow cytometry. Systemic treatment of mice with EMP after electrical denudation of the endothelium accelerated reendothelialization in vivo (29.81±2.31 vs. 50.71±9.84, p<0.01, n=12-14). Human EMP incorporation by murine ECs was confirmed in subsequent experiments using flow cutometry and immunofluorescence. In vitro experiments revealed that EMP uptake in ECs promote EC migration (82.02±15.72 vs. 37.05±19.00, p<0.01, n=6) and proliferation (160.8±24.67 vs. 100.0±26.86, n=6), both critical steps in endothelial repair. In order to dissect the underlying mechanisms, Taqman microRNA-array was performed and microRNA (miR)-126 was identified as the predominantly expressed miR in EMP (n=3). Following experiments demonstrated that miR-126 was transported into recipient HCAEC by EMP and functionally regulated the target protein sprouty-related, EVH1 domain containing 1 (Spred1). Genetic downregulation of Spred1 in HCAEC increased EC migration and proliferation capacity. Knockdown of miR-126 in EMP abrogated EMP-mediated effects on HCAEC migration and proliferation in vitro and reendothelialization in vivo, confirming the crucial role of miR-126 in EMP-mediated endothelial regeneration. Next, we tested whether EMP generated under pathological hyperglycaemic conditions (defined as injured EMP, (iEMP)) differed in miR expression and function from EMP. Interestingly, Taqman microRNA-array analysis revelaed that miR-126 was the strongest regulated miR between iEMP and EMP (n=3). Subsequently, iEMP derived from glucose-treated ECs showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Conclusion: Endothelial microparticles promote vascular endothelial repair by delivering functional microRNA-126 into recipient cells. This leads to downregulation of target protein Spred1 and improves migratory and proliferative EC capacity with subsequent improvement of EC repair. In pathological hyperglycaemic conditions, EMP-mediated miR-126 induced EC repair is altered.

Importance of tumor suppressor gene p53-mediated endothelial cell apoptosis for cardiac angiogenesis and hypertrophy

Cardiac hypertrophy initially develops as adaptive response of the heart to increased workload, but may progress to organ dysfunction if continued over the long term. Previous studies suggested that the rarefication of cardiac endothelial cells may be causally involved in the development of heart failure. The aim of this study was to examine whether prevention of endothelial cell apoptosis, achieved by deletion of the tumor suppressor gene p53, may improve cardiac remodelling in response to increased afterload and prevent or postpone the transition to heart failure. Mice with endothelial cell-specific deletion of p53 (End.p53-KO) were generated by crossing p53flox/flox mice with mice expressing Cre recombinase under control of the tamoxifen-inducible, endothelial cell-specific promoter Tie2 (Tie2.ERT.Cre). Cardiac hypertrophy was induced using the transaortic constriction (TAC) model. In control mice, cardiac hypertrophy was accompanied by increased p53 protein expression (P<0.05 vs. sham) and elevated levels of apoptosis (P<0.01), as shown by TUNEL and activated caspase-3 staining. In contrast, reduced apoptotic cell numbers were detected in hearts of End.p53-KO mice after TAC (P<0.01 vs. controls). Moreover, End.p53-KO mice exhibited increased cardiac capillary density (P<0.001) and higher number of lectin-perfused blood vessels (P=0.0002). Endothelial p53 deletion also improved extracardiac angiogenesis in the unilateral hindlimb ischaemia model and was associated with enhanced reperfusion (P<0.001) and increased capillary density (P<0.001). In vitro analyses confirmed that inhibition or transient reduction of p53, achieved by preincubation of human microvascular endothelial cells with pifithrin-α or p53-specific small interfering RNAs, promoted endothelial sprouting (P<0.05). In addition to improved cardiac angiogenesis, hearts of End.p53-KO mice exhibited less interstitial fibrosis compared to those of controls (P<0.001) and expressed lower mRNA levels of p53-regulated target genes involved in extracellular matrix remodelling (including collagen, CTGF or PAI-1). Importantly, echocardiographical measurements revealed less severe fractional shortening reduction (P<0.05 vs. controls) and better survival in End.p53-KO mice 20 weeks after TAC. Taken together, our findings reveal that accumulation of p53 in endothelial cells contributes to the rarefication of blood vessels and the development of fibrosis during cardiac hypertrophy and that apoptotic endothelial cell death may be involved in the development of heart failure in response to chronic elevations of cardiac afterload.

Natural antibodies of newborns recognize oxidative stress-related malondialdehyde acetaldehyde adducts on apoptotic cells and atherosclerotic plaques

Malondialdehyde acetaldehyde (MAA) adducts are generated under oxidative stress and shown to be highly immunogenic. Our aim was to investigate the recognition of MAA adducts by human natural antibodies in newborns before or at the time of full-term pregnancy. Plasma samples of pre-term (n = 11) and full-term (n = 36) newborns were enriched in specific IgM binding to MAA adducts compared with the maternal plasma IgM levels. Umbilical cord blood lymphocyte phage display library was generated to clone Fabs that specifically recognized MAA adducts without cross-reactivity to malondialdehyde. Fab clones from the antibody libraries of the pre-term and full-term newborns showed high sequence homology to the germline genes encoding the variable regions of antibodies, confirming that these Fabs represented the natural antibody repertoire of human fetuses. The MAA-specific umbilical cord blood Fabs bound to apoptotic human endothelial cells and the binding was efficiently competed with MAA adducts. The MAA-specific Fabs also recognized epitopes on advanced atherosclerotic lesions, and the uptake of infrared (IR)-labeled MAA-low-density lipoprotein by mouse J774A.1 macrophages was significantly reduced in the presence of these Fabs. In conclusion, MAA adducts were identified as one of the major antigenic targets for human natural antibodies already before the time of birth. MAA-specific natural antibodies are suggested to regulate apoptotic cell clearance starting from fetal development and to participate in the immunomodulation of atherosclerosis development during adulthood.

The role of telomere shortening in the development and progression of COPD

The prevalence and incidence of COPD increase with age in all populations studied. These associations suggest that normal aging could contribute to the pathogenesis of COPD. Telomeres are DNA structures at the end of chromosomes which protect DNA from degradation and unneeded recombination events. Telomere length decreases with age. At a critical length cells undergo senescence which results in changes in cell morphology, gene activity, and cytokine production. Senescent cells eventually undergo programmed cell death (apoptosis) and drop out of tissue structures. Telomeres from lung tissue and circulating leukocytes are shorter in patients with COPD. Lung biopsy specimens indicate that patients with COPD have increased numbers of apoptotic epithelial and endothelial cells. This information suggests that normal aging or accelerated aging or senescence induced by oxidant injury and/or inflammation contributes to the development of COPD. Longitudinal studies suggest that patients with shorter telomere have increased all cause and cancer mortality. Therefore, telomere attrition is associated with important consequences in these patients. In addition, animal studies suggest that patients with shorter telomeres are at increased risk of developing COPD. These observations raise the possibility that drugs designed to increase telomere length or to prevent telomere attrition might slow the progression of COPD. However, very careful studies with animal models of COPD and of malignancy are needed to determine benefits and to make certain that this genetic manipulation does not increase the risk of malignancy.

Differences in the released endothelial microparticle subtypes between human pulmonary microvascular endothelial cells and aortic endothelial cells in vitro

ABSTRACTCirculating endothelial microparticles (EMPs) are membrane vesicles that are shed into the blood stream from activated or apoptotic endothelial cells. We previously reported that circulating EMP numbers significantly increased in stable chronic obstructive pulmonary disease (COPD) patients and during exacerbation compared with healthy control subjects. However, different types of circulating EMPs with distinct time profiles were detectable during exacerbations. We hypothesized that the released EMP subtypes correlated with differences in the inflammatory stimuli and the endothelial cell type. We compared the EMP subtypes from human aortic endothelial cells (Aortic ECs) and human lung microvascular endothelial cells (Pulmonary microvascular ECs) released in response to various stimuli, including proinflammatory cytokines (TNFα), oxidative stress (H2O2), and cigarette smoke extracts (CSE) in vitro. We defined circulating EMPs by the expression of endothelial antigens: CD144+ MPs (VE-cadherin EMPs), CD31+/CD41− MPs (PECAM EMPs), CD62E+ MPs (E-selectin EMPs), and CD146+ MPs (MCAM EMPs). E-selectin EMPs were released from both pulmonary microvascular and aortic ECs in response to TNFα but not to H2O2 or CSE stimulation. The amount of MCAM EMPs released from pulmonary microvascular ECs differed significantly between the cells stimulated with H2O2 and those stimulated with CSE. VE-cadherin EMPs were only released from aortic ECs, whereas PECAM EMPs were released exclusively from pulmonary microvascular ECs. The EMP subtypes released differ in vitro among TNFα, H2O2, and CSE stimulation as well as between pulmonary microvascular and aortic ECs. The differences in circulating EMP subtypes may reflect a condition or site of endothelial injury and may serve as markers for endothelial damage in COPD patients.

Antiperlecan Antibodies Are Novel Accelerators of Immune-Mediated Vascular Injury

Acute vascular rejection (AVR) is characterized by immune-mediated vascular injury and heightened endothelial cell (EC) apoptosis. We reported previously that apoptotic ECs release a bioactive C-terminal fragment of perlecan referred to as LG3. Here, we tested the possibility that LG3 behaves as a neoantigen, fuelling the production of anti-LG3 antibodies of potential importance in regulating allograft vascular injury. We performed a case-control study in which we compared anti-LG3 IgG titers in kidney transplant recipients with AVR (n=15) versus those with acute tubulo-interstitial rejection (ATIR) (n=15) or stable graft function (n=30). Patients who experienced AVR had elevated anti-LG3 titers pre and posttransplantation compared to subjects with ATIR or stable graft function (p<0.05 for both mediators). Elevated pretransplant anti-LG3 titers (OR: 4.62, 95% CI: 1.08-19.72) and pretransplant donor-specific antibodies (DSA) (OR 4.79, 95% CI: 1.03-22.19) were both independently associated with AVR. To address the functional role of anti-LG3 antibodies in AVR, we turned to passive transfer of anti-LG3 antibodies in an animal model of vascular rejection based on orthotopic aortic transplantation between fully MHC-mismatched mice. Neointima formation, C4d deposition and allograft inflammation were significantly increased in recipients of an ischemic aortic allograft passively transferred with anti-LG3 antibodies. Collectively, these data identify anti-LG3 antibodies as novel accelerators of immune-mediated vascular injury and obliterative remodeling.

Hypoglycemia Reduces Vascular Endothelial Growth Factor A Production by Pancreatic Beta Cells as a Regulator of Beta Cell Mass

VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.