Relationship of Endothelial Microparticles to Obesity and Cardiovascular Disease Risk in Children and Adolescents.

Background Microparticles and endothelial microparticles (EMPs) are implicated in accelerating cardiovascular disease (CVD); however, data in pediatrics are limited. We examined the relationship of microparticles and EMPs with adiposity and subclinical CVD risk measures in a pediatric population to determine their potential as biomarkers of CVD risk. Methods and Results A cross-sectional study of youth (n=280; ages 8-20 years) with a range of body mass index categories was used. Microparticles, EMPs, and activated EMPs were measured by flow cytometry. %Body fat and %visceral adipose tissue were measured by dual X-ray absorptiometry. Measures of arterial stiffness and vascular wall structure were obtained. Linear regression (with log-transformed outcomes) and logistic regression were used to evaluate associations and all results were exponentiated. Youth with overweight/obesity and severe obesity had 2.50 (95% CI, 1.56-4.01) and 3.42 (95% CI, 2.15-5.43) times the geometric means of the total number of microparticles, respectively, compared with those with normal weight. Youth with overweight/obesity and severe obesity had 1.97 (95% CI, 1.09-3.55) and 2.34 (95% CI, 1.31-4.19) times the geometric means of the total number of EMPs, respectively, compared with those with normal weight. There were positive associations between the levels of both microparticles and EMPs with higher adiposity measures and poor CVD risk measures. Youth with higher adiposity showed 1.84 times the odds of having high levels of activated EMPs (%) (odds ratio, 1.84; 95% CI, 1.08-3.14) compared with those with normal weight. Conclusions Levels of microparticles, EMPs, and activated EMPs were positively associated with adiposity and poor subclinical CVD risk in a pediatric population.

Multiple inducers of endothelial NOS (eNOS) dysfunction in sickle cell disease.

A characteristic aspect of the robust, systemic inflammatory state in sickle cell disease is dysfunction of endothelial nitric oxide synthase (eNOS). We identify 10 aberrant endothelial cell inputs, present in the specific sickle context, that are known to have the ability to cause eNOS dysfunction. These are: endothelial arginase depletion, asymmetric dimethylarginine, complement activation, endothelial glycocalyx degradation, free fatty acids, inflammatory mediators, microparticles, oxidized low density lipoproteins, reactive oxygen species, and Toll-like receptor 4 signaling ligands. The effect of true eNOS dysfunction on clinical testing using flow-mediated dilation can be simulated by two known examples of endothelial dysfunction mimicry (hemoglobin consumption of NO; and oxidation of smooth muscle cell soluble guanylate cyclase). This lends ambiguity to interpretation of such clinical testing. The presence of these multiple perturbing factors argues that a therapeutic approach targeting only a single injurious endothelial input (or either example of mimicry) would not be sufficiently efficacious. This would seem to argue for identifying therapeutics that directly protect eNOS function or application of multiple therapeutic approaches.

Targeting the AnxA1/Fpr2/ALX pathway regulates neutrophil function, promoting thromboinflammation resolution in sickle cell disease.

Neutrophils play a crucial role in the intertwined processes of thrombosis and inflammation. An altered neutrophil phenotype may contribute to inadequate resolution, which is known to be a major pathophysiological contributor of thromboinflammatory conditions such as sickle cell disease (SCD). The endogenous protein annexin A1 (AnxA1) facilitates inflammation resolution via formyl peptide receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting the neutrophil-dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of the FPR2/ALX (a fundamental receptor involved in resolution) pathway. We found direct evidence that neutrophils with SCD phenotype play a key role in contributing to thromboinflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) to enable resolution. We present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thromboinflammatory conditions such as SCD.

Relationship of Circulating Endothelial Cells With Obesity and Cardiometabolic Risk Factors in Children and Adolescents.

Background Circulating endothelial cells (CECs) reflect early changes in endothelial health; however, the degree to which CEC number and activation is related to adiposity and cardiovascular risk factors in youth is not well described. Methods and Results Youth in this study (N=271; aged 8-20 years) were classified into normal weight (body mass index [BMI] percentage <85th; n=114), obesity (BMI percentage ≥95th to <120% of the 95th; n=63), and severe obesity (BMI percentage ≥120% of the 95th; n=94) catagories. CEC enumeration was determined using immunohistochemical examination of buffy coat smears and activated CEC (percentage of vascular cell adhesion molecule-1 expression) was assessed using immunofluorescent staining. Cardiovascular risk factors included measures of body composition, blood pressure, glucose, insulin, lipid profile, C-reactive protein, leptin, adiponectin, oxidized low-density lipoprotein cholesterol, carotid artery intima-media thickness, and pulse wave velocity. Linear regression models examined associations between CEC number and activation with BMI and cardiovascular risk factors. CEC number did not differ among BMI classes (P>0.05). Youth with severe obesity had a higher degree of CEC activation compared with normal weight youth (8.3%; 95% CI, 1.1-15.6 [P=0.024]). Higher CEC number was associated with greater body fat percentage (0.02 per percentage; 95% CI, 0.00-0.03 [P=0.020]) and systolic blood pressure percentile (0.01 per percentage; 95% CI, 0.00-0.01 [P=0.035]). Higher degree of CEC activation was associated with greater visceral adipose tissue (5.7% per kg; 95% CI, 0.4-10.9 [P=0.034]) and non-high-density lipoprotein cholesterol (0.11% per mg/dL; 95% CI, 0.01-0.21 [P=0.039]). Conclusions Methods of CEC quantification are associated with adiposity and cardiometabolic risk factors and may potentially reflect accelerated atherosclerosis as early as childhood.

Abnormal Endothelial Gene Expression Associated With Early Coronary Atherosclerosis.

Background We examined feasibility of a unique approach towards gaining insight into heritable risk for early atherosclerosis: surveying gene expression by endothelial cells from living subjects. Methods and Results Subjects aged <50 years (mean age, 37; range, 22-49) without obstructive coronary artery disease underwent coronary reactivity testing that identified them as having normal or abnormal coronary endothelial function. Cultures of Blood Outgrowth Endothelial Cells (BOEC) from 6 normal and 13 abnormal subjects passed rigorous quality control and were used for microarray assessment of gene expression. Of 9 genes differentially expressed at false discovery rate <0.1%, we here focus upon abnormal subjects having elevated expression of HMGB1 (high mobility group box 1) which we unexpectedly found to be linked to low LAMC1 (laminin gamma 1) expression. This linkage was corroborated by 3 of our past studies and confirmed bio-functionally. Compared with normal BOEC, abnormal BOEC released 13±3-fold more HMGB1 in response to lipopolysaccharide; and they deposited one tenth as much LAMC1 into collagen subendothelial matrix during culture. Clinical follow-up data are provided for 4 normal subjects (followed 13.4±0.1 year) and for 12 abnormal subjects (followed 9.1±4.5 years). Conclusions The known pathogenic effects of high-HMGB1 and low-LAMC1 predict that the combination would biologically converge upon the focal adhesion complex, to the detriment of endothelial shear responsiveness. This gene expression pattern may comprise a heritable risk state that promotes early coronary atherosclerosis. If so, the testing could be applied even in childhood, enabling early intervention. This approach offers a way to bridge the information gap between genetics and clinical phenotype.

Blood Outgrowth Endothelial Cells as a Cellular Carrier for Oncolytic Vesicular Stomatitis Virus Expressing Interferon-ß in Preclinical Models of Non-Small Cell Lung Cancer.

Oncolytic viruses have demonstrated efficacy in numerous tumor models including non-small cell lung cancer (NSCLC). One limitation of viral therapy for metastatic lung cancer is that systemic administration can be hindered by complement and antiviral immunity. Thus, we investigated whether ex vivo-infected blood outgrowth endothelial cells (BOECs) could be used to deliver VSV-IFNß in preclinical models of NSCLC. BOECs were obtained from human donors or C57/Bl6 mice. VSV was engineered to produce GFP or IFNß. Human and murine BOECs could be infected by VSV-GFP and VSV-IFNß. Infected BOECs resulted in killing of NSCLC cells in vitro and shielded VSV-IFNß from antibody neutralization. Mouse BOECs localized to lungs of mice bearing syngeneic LM2 lung tumors, and infected murine BOECs reduced tumor burden in this model. In an immune-deficient A549 xenograft model, mice treated with VSV-IFNß-infected human BOECs exhibited superior antitumor activity and survival of mice (n = 10, P < .05 compared to VSV-IFNß alone). We conclude that BOECs can be used as a carrier for delivery of oncolytic VSV-IFNß. This may be an effective strategy for clinical translation of oncolytic virotherapy for patients with metastatic NSCLC.

The multifaceted role of ischemia/reperfusion in sickle cell anemia.

Sickle cell anemia is a unique disease dominated by hemolytic anemia and vaso-occlusive events. The latter trigger a version of ischemia/reperfusion (I/R) pathobiology that is singular in its origin, cyclicity, complexity, instability, perpetuity, and breadth of clinical consequences. Specific clinical features are probably attributable to local I/R injury (e.g., stroke syndromes) or remote organ injury (e.g., acute chest syndrome) or the systematization of inflammation (e.g., multifocal arteriopathy). Indeed, by fashioning an underlying template of endothelial dysfunction and vulnerability, the robust inflammatory systematization no doubt contributes to all sickle pathology. In this Review, we highlight I/R-targeting therapeutics shown to improve microvascular blood flow in sickle transgenic mice undergoing I/R, and we suggest how such insights might be translated into human therapeutic strategies.

Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/- . These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.

Reproducibility of endothelial microparticles in children and adolescents.

Aim: We assessed reproducibility of endothelial microparticles (EMPs) enumeration among youth. Methods & results: Four microparticle (MP) indices - total MP per microliter platelet free plasma (PFP), total EMPs per microliter PFP, percent activated EMPs and percent lactadherin positive (LACT[+]) of total EMPs - were measured at two visits (baseline and 7 ± 3 days follow-up) to determine reproducibility overall and by obesity status. We examined CD31+ or CD144+ with CD41-EMP events of size 0.3-1.0 µm. No statistically significant differences were observed between visits for any of the four MP indices. The within-participant and between-participant coefficient of variation was acceptable (range: 1.13-2.37) with good intraclass-correlation coefficient for all indices except total MP per microliter (range: 0.10-1.00). Conclusion: Total EMPs per microliter PFP, percent-activated EMPs and percent LACT(+) of total EMPs are reproducible among youth.

Blood outgrowth endothelial cells overexpressing eNOS mitigate pulmonary hypertension in rats: a unique carrier cell enabling autologous cell-based gene therapy.

We have investigated a unique cell type, blood outgrowth endothelial cells (BOEC), as a cell-based gene therapy approach to pulmonary hypertension. BOEC are bona fide endothelial cells, obtained from peripheral blood, that can be expanded to vast numbers, and are amenable to both cryopreservation and genetic modification. We established primary cultures of rat BOEC and genetically altered them to over-express human eNOS plus green fluorescent protein (rBOEC/eNOS) or to express GFP only (rBOEC/GFP). We gave monocrotaline to rats on day 0, and they developed severe pulmonary hypertension. As a Prevention model, we infused saline or rBOEC/GFP or rBOEC/eNOS on day 3, and then examined endpoints on day 24. The rBOEC/eNOS recipients developed elevated NOx (serum and lung) and less severe: elevation of right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary arteriolar muscularization and loss of alveolar density. As an Intervention model, we waited until day 21 to give the test infusions, and we examined endpoints on day 35. The rBOEC/eNOS recipients again developed elevated NOx and manifested the same improvements. Indeed, rBOEC/eNOS infusion not only prevented worsening of RVSP but also partially reversed established arteriolar muscularization. These data suggest that BOEC may be useful as a carrier cell for genetic strategies targeting pulmonary hypertension. Their properties render BOEC amenable to preclinical and scale-up studies, available for autologous therapies, and tolerant of modification and storage for potential future use in patients at risk for PAH, eg, as defined by genetics or medical condition.

Morphine promotes neovascularizing retinopathy in sickle transgeneic mice.

Neovascularizing retinopathy is a significant complication of sickle cell disease (SCD), occurring more frequently in HbSC than HbSS disease. This risk difference is concordant with a divergence of angiogenesis risk, as identified by levels of pro- vs anti-angiogenic factors in the sickle patient's blood. Because our prior studies documented that morphine promotes angiogenesis in both malignancy and wound healing, we tested whether chronic opioid treatment would promote retinopathy in NY1DD sickle transgenic mice. After 10 to 15 months of treatment, sickle mice treated with morphine developed neovascularizing retinopathy to a far greater extent than either of the controls (sickle mice treated with saline and wild-type mice treated identically with morphine). Our dissection of the mechanistic linkage between morphine and retinopathy revealed a complex interplay among morphine engagement with its µ opioid receptor (MOR) on retinal endothelial cells (RECs); morphine-induced production of tumor necrosis factor α and interleukin-6 (IL-6), causing increased expression of both MOR and vascular endothelial growth factor receptor 2 (VEGFR2) on RECs; morphine/MOR engagement transactivating VEGFR2; and convergence of MOR, VEGFR2, and IL-6 activation on JAK/STAT3-dependent REC proliferation and angiogenesis. In the NY1DD mice, the result was increased angiogenesis, seen as neovascularizing retinopathy, similar to the retinal pathology occurring in humans with SCD. Therefore, we conclude that chronic opioid exposure, superimposed on the already angiogenic sickle milieu, might enhance risk for retinopathy. These results provide an additional reason for development and application of opioid alternatives for pain control in SCD.

Arterial elasticity as a risk factor for early cardiovascular disease among testicular cancer survivors treated with platinum-based chemotherapy: a cross-sectional pilot study.

PURPOSE: Testicular cancer survivors who have received platinum-based chemotherapy are at risk for premature cardiovascular disease. The etiology of this risk is not well understood. This pilot study explores the impact of platinum-based chemotherapy on endothelial function. METHODS: Testicular cancer survivors <30 years old at the time of diagnosis who received platinum-based chemotherapy between 2002 and 2012, as well as 17 similarly aged male controls, were identified. Consented subjects underwent vascular assessment using the HDI/PulseWave CR-2000 Cardiovascular Profiling System and the Endo-PAT2000 system. Biomarkers and functional test markers were compared among cases, controls, and a group of historical controls using two sided two-sampled t-tests and Wilcoxon rank-sum tests. RESULTS: Thirteen survivors with a median age of 30.2 years and body mass index of 27.3 were enrolled, along with 17 healthy controls with a median age of 27.1 years and body mass index of 24.8. Median time from chemotherapy was 4.7 (range: 0.8-14) years. There was no statistical difference in reactive hyperemia peripheral arterial tonometry ratio between cases and controls (p = 0.574). There was no statistical difference in small or large artery elasticity between cases and controls (p = 0.086) or between cases and historical controls (p = 0.729). There was also no statistical difference in the blood levels of circulating endothelial cells, von Willebrand factor, and vascular cell adhesion molecules. There was a trend toward increased metabolic syndrome in cases (15%) as compared to recruited controls (6%), though this difference was not statistically significant (p = 0.565). CONCLUSION: Testicular cancer survivors have no clinically significant difference in endothelial function compared to controls 4 years after the completion of chemotherapy. Further research is needed to explore the secondary modifiable causes that may contribute to the risk of premature cardiovascular disease.

Vascular function in breast cancer survivors on aromatase inhibitors: a pilot study.

PURPOSE: Aromatase inhibitors (AI) have been shown to reduce breast cancer-related mortality in women with estrogen positive (ER+) breast cancer. The use of AIs, however, has been associated with higher rates of hypertension, hyperlipidemia, and cardiovascular (CV) events. METHODS: A cross-sectional study of 25 healthy postmenopausal women and 36 women with curative intent breast cancer on an AI was performed to assess endothelial dysfunction, an indicator of risk for CV events. Consented subjects underwent vascular testing using the HDI/Pulse Wave CR-2000 Cardiovascular Profiling System and the EndoPAT2000 system. RESULTS: Mean age was 61.7 and 59.6 years (cases, controls). Most subjects were Caucasian and overweight. Controls had a lower mean systolic blood pressure (128.6 mmHg vs. 116.2 mmHg, p = 0.004). Median estradiol levels were reduced in cases (2 vs. 15 pg/ml, p < 0.0001). EndoPAT ratio (0.8 vs. 2.7, p < 0.0001) was significantly reduced in cases as compared to controls. Median large artery elasticity (12.9 vs. 14.6 ml/mmHg × 10, p = 0.12) and small artery elasticity (5.2 vs. 7.0 ml/mmHg × 100, p = 0.07) were also reduced though not statistically significant. There was no correlation between use of chemotherapy, radiation therapy, type of AI, or duration of AI use and endothelial function. When adjusting for differences in blood pressure, results remained significant. CONCLUSION: Breast cancer cases on AIs have reductions in endothelial function, a predictor of adverse CV disease. IMPACT: Vascular function changes in breast cancer cases on AIs compared to postmenopausal women. Further work is needed to evaluate vascular changes over time.

A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles , and SS-BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr-1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL-1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro-inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM-1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent-and possibly dominant-role for an abnormal monocyte-TNF-endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.

Blood endothelial cells: utility from ambiguity.

In the mid-1990s, my research group began to devise a method to establish endothelial cell cultures from human peripheral blood, with an ultimate goal of examining interindividual heterogeneity of endothelial biology. The initial work, published in the JCI in 2000, described the method enabling successful attainment of blood outgrowth endothelial cells (BOEC). Truly endothelial, BOEC are progeny of a transplantable cell that originates in bone marrow, a putative endothelial progenitor. Our subsequent experimental work focused upon practical applications of BOEC: their use for gene therapy, tissue engineering, assessment of mutant gene effect, and discovery of heterogeneity in endothelial biology.

Microparticles in sickle cell anaemia: promise and pitfalls.

Blood from patients with sickle cell disease contains microparticles (MP) derived from multiple cell sources, including red cells, platelets, monocytes and endothelial cells. MPs are of great interest because of their disease associations, their status as promising biomarkers, and the intercellular communications they mediate. To illustrate the likelihood of their relevance in sickle cell disease, we discuss the nature of MP, their profiling in sickle disease, some caveats relevant to their detection, their roles in supporting coagulation and the disparate influences they may exert upon the pathobiology of sickle cell disease.