Biomarkers Of Vascular Injury Research Articles

Association of MRI markers of vascular injury and neurodegeneration and cognitive decline in diverse cohorts: Findings from KHANDLE and STAR

AbstractBackgroundMRI biomarkers of vascular injury and neurodegeneration are associated with cognitive decline, but relatively little is known about cognitive change in diverse populations. Our objective is to examine associations of hippocampal volume and white matter integrity on cognitive change over 3 years.MethodKHANDLE (age 65+) and STAR (age 50+) are harmonized cohorts of racially/ethnically diverse long‐term members of Kaiser Permanente Northern California. A random sub‐sample of participants received neuroimaging with 3T MRI to measure regional brain volumes and white matter integrity. Hippocampal volume (cm3), log white matter hyperintensities (log‐WMH) (cm3), free water (FW) (cm3), and fractional anisotropy (FA) MRI measures were normalized on intracranial volume and age at scan. Cognitive decline was assessed across three waves using the Spanish English Neuropsychological Assessment Scale measuring executive function (EF) and verbal episodic memory (VEM). Linear mixed models allowing for random intercept and slope estimated the associations of MRI metrics and cognition adjusting for baseline age, education, gender/sex, interview mode (in‐person vs phone), and interactions of these covariates with time from baseline, to adjust for effects of covariates on both baseline cognition and cognitive change.ResultAmong 592 participants with neuroimaging, 59% were women, 47% were college educated, and 52% were Black adults (Table 1). Hippocampal volume was positively associated with baseline VEM (β = 0.268; CI = 0.153, 0.383) and EF change (β = 0.051; CI = 0.009, 0.094) (Table 2, Plot 1). FW was negatively associated with baseline EF (β = ‐6.561; CI = ‐10.758, ‐2.364), baseline VEM (β = ‐5.557; CI = ‐10.03, ‐1.084) and EF decline (β = ‐1.667; CI = ‐3.268, ‐0.066). FA was positively associated with baseline EF (β = 6.222; CI = 3.217, 9.228) and baseline VEM (β = 6.944; CI = 3.768, 10.119). Log‐WMH was negatively associated with baseline EF (β = ‐0.068; CI = ‐0.116, ‐0.020) and VEM (β = ‐0.082; CI = ‐0.132, ‐0.032).ConclusionAll MRI markers were associated with baseline cognition, but only hippocampal volume and FW were associated with cognitive change. MRI measures of neurodegeneration and white matter integrity may be predictive of cognition and short‐term decline in diverse groups of older adults. Future analyses will examine associations with cognitive impairment.

Thrombomodulin Protects Against Cell-Free Hemoglobin Mediated Acute Kidney Injury in Sickle Cell Anemia

Thrombomodulin (TM) is a transmembrane protein that plays a pivotal role in maintaining microvascular health. Functions of TM include 1) binding thrombin and inhibiting its interaction with fibrinogen, 2) augmenting protein C function, and 3) down-regulating complement activation. Reduced TM function has been implicated in several thrombotic microangiopathies, including atypical hemolytic uremic syndrome and disseminated intravascular coagulation (DIC) (PMID 29866818). Previous in vitro studies have demonstrated that cell-free hemoglobin (Hb) mediates loss of TM from the endothelial surface which, in turn, is associated with a higher risk of kidney injury in patients with sickle cell disease (PMID 34929051). Infusion of intact, soluble TM may restore TM's function in the microvasculature, as observed in critically ill patients with DIC (PMID 17059423). In transgenic sickle mice (Townes model, Jackson Laboratory; Bar Harbor, Maine), we investigated whether 1) cell-free Hb reduces TM function leading to glomerular endothelial and kidney injury and 2) if infusion of intact, soluble TM (Asahi Kasei Pharma ©; Tokyo, Japan) can restore TM function and reduce the toxic effects of cell-free Hb in the kidney. Sickle mice (4 male, 4 females per condition) were challenged with cell-free Hb (0.24g/kg intravenous [iv]) or normal saline followed 2 hours later by either infusion of TM (5mg/kg subcutaneous, 1mg/kg iv) or normal saline. Blood and urine samples were collected 24 hours after the challenge and the mice were sacrificed for histopathologic evaluation. In a separate set of similar experiments, sickle mice (3 males, 3 females per condition) received iv microbubbles (Vevo MicroMarker TM Contrast) 24 hours after the challenge to assess renal cortical blood flow by contrast-enhanced ultrasound (Vevo2100 system). Mean ± standard error of mean values are provided. Comparisons between Hb-only and Hb+TM conditions were made using the Mann-Whitney test. Vasculopathy: Infusion of cell-free Hb resulted in decreased TM antithrombotic function within the glomerulus; infusion of TM 2 hours after the challenge restored these functions (fibrinogen relative fluorescent intensity [RFI]: control, 24.2 ± 0.9; Hb-only, 40.6 ± 1.7; Hb+TM: 26.8 ± 1.6) and anti-complement function (C3 RFI: control, 12.9 ± 0.4; Hb-only, 19.3 ± 1.4; Hb+TM, 14.1 ± 0.5) (P≤0.007). Circulating vascular injury biomarkers rose in the cell-free Hb challenged sickle mice and this was abated with TM rescue for p-selectin (control, 271 ± 25 ng/mL; Hb-only, 533 ± 47 ng/mL; Hb+TM, 356 ± 34 ng/mL), VEGF (control, 59 ± 4 pg/mL; Hb-only, 93 ± 7 pg/mL; Hb+TM 69 ± 3 pg/mL), and vWF (control: 91 ± 9 ng/mL, Hb-only: 299 ± 31 ng/mL, Hb+TM: 183 ± 22 ng/mL) (P≤0.01). Kidney Damage: Urine biomarkers of tubular injury increased after the cell-free Hb challenge and improved with TM (KIM-1: control, 317 ± 66 pg/day; Hb-only, 1201 ± 142 pg/day; Hb+TM, 638 ± 78 pg/day; P=0.003) (NGAL: control, 234 ± 34 ng/day; Hb-only, 1624 ± 145 ng/day; Hb+TM, 767 ± 118 ng/day; P=0.003). An elevation in serum creatinine concentration is used to define acute kidney injury. The cell-free Hb challenge led to an acute rise in serum creatinine while the TM rescued mice had serum creatinine concentrations that were significantly lower than the Hb-only challenged mice (P=0.03) and similar to the control conditions (Figure 1A). Preliminary testing of TM administered 24 hours after the cell-free Hb challenge has also led to kidney protective effects as assessed by serum creatinine, p-selectin, and vWF (P < 0.05). Cortical Blood Flow: Peak enhancement, a measure of relative blood volume, was reduced in the cell-free Hb challenged mice compared to control conditions by contrast-enhanced ultrasound. The TM-rescued mice had improved peak enhancement compared to the Hb-only treated mice (P=0.03) and similar to control conditions (Figure 1B). In conclusion, cell-free Hb reduces TM function resulting in increased vascular and kidney injury and decreased cortical blood flow. Infusion of soluble TM 2 hours after the cell-free Hb challenge restores TM function, protects against vascular and kidney injury, and preserves cortical blood flow. Our data highlights TM as a potential candidate therapy to preserve vascular function and abate kidney injury in patients with sickle cell disease during severe vaso-occlusive or hyperhemolytic episodes, when concentrations of cell-free Hb increase several fold.

S-19-2: EXTRACELLULAR VESICLES: BIOMARKERS AND BEYOND

Extracellular vesicles (EVs) are small membrane-enclosed particles that are released by cells into the extracellular environment. They are increasingly recognized as key participants in intercellular signaling where they may either promote or prevent vascular injury depending on the context. Moreover, as their molecular composition is reflective of their cell/tissue of origin, they are increasingly employed as biomarkers of vascular injury. This presentation will discuss key work from our laboratory and others focused on EVs in hypertension and related cardiovascular diseases such as diabetes.

Reduced circulating BMP9 and pBMP10 in hospitalized COVID-19 patients.

Similar to other causes of acute respiratory distress syndrome, coronavirus disease 2019 (COVID-19) is characterized by the aberrant expression of vascular injury biomarkers. We present the first report that circulating plasma bone morphogenetic proteins (BMPs), BMP9 and pBMP10, involved in vascular protection, are reduced in hospitalized patients with COVID-19.

Onion and Apple Functional Ingredients Intake Improves Antioxidant and Inflammatory Status and Vascular Injury in Obese Zucker Rats.

The objective of this study was to investigate the effects of onion and apple functional ingredients in homozygous (fa/fa) obese Zucker rats. Rodents were fed three diets: standard diet [obese control (OC) group], standard diet containing 10% onion [obese onion 10% (OO) group] and standard diet containing 10% apple [obese apple 10% (OA) group] for 8 weeks. Food intake and body weight gain were higher in obese than in lean rats. Food efficiency was lower in OO and AO groups compared with OC group. Within the obese groups, total cholesterol, LDL-cholesterol, triacylglycerols, glucose, insulin and triglyceride-glucose index were lower in OO group than in OC group, and HDL-cholesterol was higher in OO group than in OC group. In general, antioxidant activity (ABTS•+ and FRAP), antioxidant enzyme activities (CAT, SOD, GPx), GSH/GSSG ratio, nitrate/nitrite and GLP-1 increased in OO and OA groups compared with OC. Oxidative stress biomarkers, namely protein carbonyls, 8-hydroxy-2′-deoxyguanosine, 8-epi-prostaglandin F2α, inflammatory and vascular injury biomarkers (PAI-1, TIMP-1, VEGF, sICAM-1, sE-Selectin, MCP-1) and leptin, were lower in OO and OA groups than in OC group. Endothelial impairment was partially reversed, and superoxide content and gene expression of NLRP3, NFKβ1 and COX2 decreased, in OO and OA groups with respect to OC group. The study demonstrates that high pressure-processed onion and apple functional ingredients administration to obese Zucker rats causes beneficial effects on metabolic health, in particular through improving food efficiency ratio; exerting pronounced lipid-lowering effects; reducing glycemia, insulinemia, and biomarkers of hepatic injury (ALT, AST); improving antioxidant, oxidative stress, inflammatory and vascular injury biomarkers, metabolic hormones, and endothelial function; and decreasing proinflammatory gene expression of NLRP3, NFKβ1 and COX2.

Endothelial biomarkers as predictors for haemodialysis need in severe leptospirosis patients (Weil's disease).

To investigate the prediction ability of vascular injury biomarkers for haemodialysis requirement in patients with severe leptospirosis. Prospective study with severe leptospirosis patients hospitalised in Fortaleza, Brazil. Blood samples were collected hospital admission to quantify vascular injury biomarkers: syndecan-1, ICAM-1, VCAM-1, angiopoietin-2 and FGF-23. Two groups were evaluated according to haemodialysis requirement during hospital stay. Twenty-seven patients were included, with a mean age of 39 ± 18 years. 88.9% were males. 53.8% needed haemodialysis and presented higher levels on hospital admission of syndecan-1 (572 [300-811] vs. 263 [106-421] ng/ml; p= 0.03), angiopoietin-2 (1.52 [0.72-2.72] vs. 0.63 [0.4-1.38] ng/ml; p= 0.01), and FGF-23 (291 [56-2031] vs. 10 [10-806] pg/ml; p= 0.021). Syndecan-1 showed significant correlation with creatinine (r= 0.546; p= 0.05) and total bilirubin levels (r= 0.534; p= 0.013) on hospital admission. Angiopoietin-2 showed significant correlation with creatinine levels (r= 0.513; p= 0.009) on hospital admission and with number of haemodialysis sessions (r= 0.406; p= 0.049). No significant correlation was found with FGF-23. Regarding prognostic performance, combined syndecan-1 and angiopoietin-2 levels had a better ability to predict haemodialysis need in patients with severe leptospirosis (AUC-ROC=0.744 [95% CI: 0.545-0.943] p= 0.035). Syndecan-1 and angiopoietin-2 were associated with haemodialysis need in patients with severe leptospirosis and may be useful to improve therapeutic approach and reduce mortality.

Inhibition of semaphorin-3a alleviates lipopolysaccharide-induced vascular injury.

Alleviating vascular injury improves the prognosis of atherosclerosis. Semaphorin-3a (Sema3A) is a special membrane-associated secreted protein with various biological properties, like pro-inflammation, anti-tumor and et al. This study aims to investigate the effects of inhibition of Sema3A on lipopolysaccharide (LPS)-induced vascular injury in mice. The mice were randomized into three groups: control, LPS, and LPS + siRNA. Mice in the combined group were given siRNA through fast tail vein injection, then LPS was injected intraperitoneally 7days later, finally the mice were euthanized 24h later. Vascular function and structure were assessed by vascular injury biomarkers and relevant stainings. LPS-induced vascular dysfunction and pathological injury were substantially improved by inhibition of Sema3A. Western blot and quantitative real-time polymerase chain reaction assays were used for investigating molecular pathways. The relevant proteins of vascular endothelial cells activation, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), increased after LPS stimulation, while these effects were reversed by inhibition of Sema3A. The levels of inflammatory cytokines (IL-1β, IL-6 and NLRP3) were upregulated after LPS stimulation, however, inhibition of Sema3A reversed it through NF-κB and MAPKs signaling pathways involvement. Moreover, inhibition of Sema3A alleviated LPS-induced oxidative stress, evidenced by a decrease in total reactive oxygen species and an increase in antioxidant protein of SOD-1. The results showed that inhibition of Sema3A protects against LPS-induced vascular injury by suppressing vascular endothelial cells activation, vascular inflammation, and vascular oxidative stress, implying that inhibition of Sema3A might be used as a therapeutic strategy for septic vascular injury or atherosclerosis.

Inflammatory profiles in plasma and cerebrospinal fluid of patients with neurosarcoidosis

MethodsCerebrospinal fluid (CSF) and plasma levels of 38 biomarkers from 20 neurosarcoidosis (NS) patients were compared to healthy controls (HC). ResultsIn CSF, 25 biomarkers were significantly elevated compared to HC: IFNγ, TNFα, TNFβ, IL-2, IL-6, IL-10, IL-12B, IL-15, IL-16, CCL2, CCL3, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26, CXCL8, CXCL10, TNFR2, VEGF-A, PIGF, SAA, VCAM1, and ICAM1.In plasma, 12 biomarkers were significantly elevated compared to HC: IFNγ, TNFα, CCL2, CCL3, CCL4, CCL17, CXCL10, VEGFR1, PIGF, SAA, VCAM1, and ICAM1. ConclusionNS patients have profoundly elevated cytokines, chemokines, vascular angiogenesis, and vascular injury biomarkers in CSF and plasma.

Best Practices for Authentication of Cell Lines to Ensure Data Reproducibility and Integrity.

Cell line misidentification and contamination are major contributors to the reproducibility crisis in academic research. Authentication of cell lines provides assurances of the data generated; however, commercially available cells are often not subjected to rigorous identification testing. In this study, commercially available cell lines underwent testing to confirm cell identity and purity. The methods reported here outline the best practices for cell line authentication. Briefly, a commercially available primary rabbit aortic endothelial cell line was purchased for the intent of producing target proteins necessary for generating species-specific recombinant antibodies. These rabbit-specific antibodies would then be utilized for the development of in-house enzyme-linked immunosorbent assays (ELISA) to evaluate blood-based biomarkers of vascular injury after total-body irradiation. To authenticate the cell line, cell identity and purity were determined by single tandem repeat (STR) testing, flow cytometry, polymerase chain reaction (PCR), and cytochrome c oxidase subunit 1 (CO1) DNA Barcoding in-house and/or through commercial vendors. Fresh cells obtained from a New Zealand White rabbit (Charles River, Wilmington, DE) were used as a positive control. The results of STR and flow cytometry analyses indicated the cells were not contaminated with human or mouse cells, and that the cells were not of endothelial origin. PCR demonstrated that cells were also not of rabbit origin, which was further confirmed by a third-party vendor. An unopened vial of cells was submitted to another vendor for CO1 DNA Barcoding analysis, which identified the cells as being purely of bovine origin. Results revealed that despite purchase through a commercial vendor, the cell line marketed as primary rabbit aortic endothelial cells were of bovine origin. Purity analysis found cells were misidentified rather than contaminated. Further investigation to determine the cell type was not performed. The most cost-effective and efficient methodology for confirming cell line identity was found to be CO1 DNA Barcoding performed by a commercial vendor.

Endothelial-T cell crosstalk contributes to vascular injury in fatty liver disease

Abstract Cardiovascular complications are often the fundamental causes of death in non-alcoholic fatty liver disease (NAFLD) patients. While there are known systemic mediators in NAFLD that may induce vascular inflammation, the mechanism of endothelial dysfunction remain understudied. In this work, we harnessed the replicative potential of blood outgrowth endothelial cells (BOECs) to develop personalized cell lines from NAFLD patients and healthy controls. Our transcriptomic analysis showed that the top interactome network enriched in NAFLD BOECs comprised of several C-C and C-X-C chemokine ligands involved in immune cell chemotaxis. We previously reported T cell infiltration in mouse model of non-alcoholic steatohepatitis, and here, we confirmed enhanced endothelial chemokine signatures in arterial histological sections. To elucidate endothelial-immune crosstalk, we performed single-cell analysis on human peripheral blood mononuclear cells and found T cell intensification in NAFLD patients compared to healthy controls. Our immunoprofiling by flow cytometry further revealed that NAFLD patients possessed higher levels CD8+ memory cells. Functionally, T cells, instead of monocytes, adhered more pronouncedly to NAFLD BOECs. In evaluating the CXCL12-CXCR4 axis in chemotaxis, CXCR4 antagonist (AMD3100) substantially modulated the migration of patient-derived CD8+ T cells towards NAFLD BOECs, which was not observed in healthy endothelial-T cell chemotaxis coculture. Finally, we validated NAFLD-associated endothelial dysfunction by enumerating two folds more circulating endothelial cells, a biomarker of vascular injury, in the blood samples of NAFLD patients than healthy controls. Our work provides insights for translation to restore blood vessel health and potentially mitigate adverse vascular events in NAFLD. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Industrial Alignment Fund Pre-Positioning grant from the Agency for Science, Technology and Research, Singapore Endothelial-T cell crosstalk in NAFLD

Effect of the Rehabilitation Program, ReStOre, on Serum Biomarkers in a Randomized Control Trial of Esophagogastric Cancer Survivors.

BackgroundThe Rehabilitation Strategies Following Esophagogastric cancer (ReStOre) randomized control trial demonstrated a significant improvement in cardiorespiratory fitness of esophagogastric cancer survivors. This follow-up, exploratory study analyzed the biological effect of exercise intervention on levels of 55 serum proteins, encompassing mediators of angiogenesis, inflammation, and vascular injury, from participants on the ReStOre trial.MethodsPatients >6 months disease free from esophagogastric cancer were randomized to usual care or the 12-week ReStOre program (exercise training, dietary counselling, and multidisciplinary education). Serum was collected at baseline (T0), post-intervention (T1), and at 3-month follow up (T2). Serum biomarkers were quantified by enzyme-linked immunosorbent assay (ELISA).ResultsThirty-seven patients participated in this study; 17 in the control arm and 20 in the intervention arm. Exercise intervention resulted in significant alterations in the level of expression of serum IP-10 (mean difference (MD): 38.02 (95% CI: 0.69 to 75.35)), IL-27 (MD: 249.48 (95% CI: 22.43 to 476.53)), and the vascular injury biomarkers, ICAM-1 (MD: 1.05 (95% CI: 1.07 to 1.66)), and VCAM-1 (MD: 1.51 (95% CI: 1.04 to 2.14)) at T1. A significant increase in eotaxin-3 (MD: 2.59 (95% CI: 0.23 to 4.96)), IL-15 (MD: 0.27 (95% CI: 0 to 0.54)) and decrease in bFGF (MD: 1.62 (95% CI: -2.99 to 0.26)) expression was observed between control and intervention cohorts at T2 (p<0.05).ConclusionsExercise intervention significantly altered the expression of a number of serum biomarkers in disease-free patients who had prior treatment for esophagogastric cancer.ImpactExercise rehabilitation causes a significant biological effect on serum biomarkers in esophagogastric cancer survivors.Clinical Trial RegistrationClinicalTrials.gov (NCT03314311).

Detection of pro angiogenic and inflammatory biomarkers in patients with CKD

Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and inflammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were significantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-β as the top pathways activated in both CKD groups. Pro-inflammatory proteins were significantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and inflammation are activated in CKD patients’ plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD.

Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation.

Numerous reports of vascular events after an initial recovery from COVID-19 form our impetus to investigate the impact of COVID-19 on vascular health of recovered patients. We found elevated levels of circulating endothelial cells (CECs), a biomarker of vascular injury, in COVID-19 convalescents compared to healthy controls. In particular, those with pre-existing conditions (e.g., hypertension, diabetes) had more pronounced endothelial activation hallmarks than non-COVID-19 patients with matched cardiovascular risk. Several proinflammatory and activated T lymphocyte-associated cytokines sustained from acute infection to recovery phase, which correlated positively with CEC measures, implicating cytokine-driven endothelial dysfunction. Notably, we found higher frequency of effector T cells in our COVID-19 convalescents compared to healthy controls. The activation markers detected on CECs mapped to counter receptors found primarily on cytotoxic CD8+ T cells, raising the possibility of cytotoxic effector cells targeting activated endothelial cells. Clinical trials in preventive therapy for post-COVID-19 vascular complications may be needed.

Human Cytomegalovirus Infections Are Associated With Elevated Biomarkers of Vascular Injury.

Background: Human cytomegalovirus (HCMV) infects ~50% of adults in the United States. HCMV infections may cause vascular inflammation leading to cardiovascular disease, but the existing evidence is inconsistent.Objective: We investigated demographic predictors of HCMV infection and explored associations between HCMV infection status, the intensity of anti-HCMV Immunoglobulin G (IgG) antibody response, and biomarkers of inflammation and endothelial function which are known predictors of cardiovascular disease.Methods: We conducted a cross-sectional study of 694 adults residing in the Raleigh-Durham-Chapel Hill, NC metropolitan area. Serum samples were tested for IgG antibody response to HCMV, and for biomarkers of vascular injury including soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), C-reactive protein (CRP), and serum amyloid A (SAA). Associations between HCMV and biomarker levels were analyzed using two approaches with HCMV serostatus modeled as a binary variable and as an ordinal variable with five categories comprised of seronegative individuals and quartiles of anti-HCMV antibody responses in seropositive individuals.Results: HCMV seroprevalence in the study population was 56%. Increased body mass index, increased age, female gender, racial/ethnic minority status, and current smoking were significantly associated with HCMV seropositivity in a multivariate regression analysis. HCMV seropositivity was also associated with 9% (95% confidence interval 4–15%) and 20% (0.3–44%) increases in median levels of sICAM-1 and CRP, respectively, after adjusting for covariates. The association between HCMV seropositivity and median levels of sVCAM-1 and SAA were positive but not statistically significant. Significant positive associations were observed between the intensity of anti-HCMV IgG responses and levels of sICAM-1 and sVCAM-1 (p-values 0.0008 and 0.04 for linear trend, respectively). To our knowledge, this is the first epidemiological study to show a relationship between anti-HCMV IgG responses and vascular injury biomarkers sICAM-1 and sVCAM-1 in the general population.Conclusion: HCMV infections are associated with vascular injury and inflammation biomarkers in adult residents of North Carolina.

Vascular injury biomarkers and stroke risk: A population-based study.

Because little is known about associations between biomarkers of vascular injury and stroke risk, we evaluated associations between plasma concentrations of 6 novel biomarkers of vascular injury and stroke risk in a population-based study. A case-cohort subset of EPIC-Heidelberg (European Prospective Investigation for Cancer and Nutrition-Heidelberg) including incident stroke cases (n = 335) and a random subcohort (n = 2,418) was selected. Concentrations of intercellular adhesion molecule 3 (ICAM3), soluble E-selectin and P-selectin, soluble thrombomodulin (sTM), thrombopoietin, and glycoprotein IIb/IIIa were measured in baseline plasma samples. Weighted Cox regression analyses were used to assess associations between biomarker levels and stroke risk. Median follow-up in the subcohort and among cases was 9.8 (range, 0.1-12.5) years and 6.2 (range, 0.01-12.1) years, respectively. ICAM3 levels were associated with increased risk of incident stroke after multivariable adjustment (hazard ratio, highest vs lowest quartile: 1.64 [95% confidence interval, 1.15-2.32]; p linear trend < 0.001). This association was more apparent for ischemic (1.65 [1.12-2.45]; p linear trend < 0.01) than for hemorrhagic stroke (1.29 [0.60-2.78]; p linear trend = 0.3). We further observed a borderline significant trend for a positive association between sTM and overall stroke risk (1.47 [0.99-2.19]; p linear trend = 0.05). In this population-based study, circulating levels of ICAM3, an adhesion molecule shed by leukocytes, were associated with increased risk of incident stroke. Further mechanistic studies are needed to elucidate the pathophysiology underlying this association. This study provides Class II evidence that plasma levels of ICAM3 are associated with increased stroke risk.

Suppression of type 2 diabetes mellitus-induced aortic ultrastructural alterations in rats by insulin: an association of vascular injury biomarkers

ABSTRACT Diabetes represents a major public health problem and an estimated 70% of people with diabetes die of cardiovascular complications. The protective effect of insulin treatment against ultrastructural damage to the tunica intima and tunica media of the aorta induced by type 2 diabetes mellitus (T2DM) has not been investigated before using transmission electron microscopy (TEM). Therefore, we induced T2DM in rats using high fat diet and streptozotocin (50 mg/kg) and administered insulin daily by i.v injection for 8 weeks to the treatment group. Whereas, the T2DM control group were left untreated for the duration of the experiment. A comparison was also made between the effect of insulin on aortic tissue and the blood level of biomarkers of vascular injury, inflammation, and oxidative stress. T2DM induced profound ultrastructural damage to the aortic endothelium and vascular smooth muscle cells, which were substantially protected with insulin. Furthermore, insulin returned blood sugar to a control level and significantly (p < .05) inhibited diabetic up-regulation of endothelial and leukocyte intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), endothelial cell adhesion molecules, P-selectin and E-selectin, tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), and malondialdehyde (MDA). Furthermore, insulin augmented the blood level of the anti-oxidant enzyme superoxide dismutase (SOD). We conclude that in a rat model of T2DM, insulin treatment substantially reduces aortic injury secondary to T2DM for a period of 8 weeks, possibly due to the inhibition of hyperglycemia, vascular activation, inflammation, and oxidative stress.

Complement fragments are biomarkers of antibody-mediated endothelial injury

Antibody-mediated rejection (AbMR) adversely affects long-term graft survival in kidney transplantation. Currently, the diagnosis of AbMR requires a kidney biopsy, and detection of complement C4d deposition in the allograft is one of the diagnostic criteria. Complement activation also generates several soluble fragments which could potentially provide non-invasive biomarkers of the process. Furthermore, microvesicles released into the plasma from injured cells can serve as biomarkers of vascular injury. To explore whether soluble complement fragments or complement fragments bound to endothelial microvesicles can be used to non-invasively detect AbMR, we developed an in vitro model in which human endothelial cells were exposed to anti-HLA antibodies and complement sufficient serum. We found that complement fragments C4a and sC5b-9 were increased in the supernatants of cells exposed to complement-sufficient serum compared to cells treated complement-deficient serum. Furthermore, complement activation on the cell surface was associated with the release of microvesicles bearing C4 and C3 fragments. We next measured these analytes in plasma from kidney transplant recipients with biopsy-proven acute AbMR (n = 9) and compared the results with those from transplant recipients who also had impaired allograft function but who did not have AbMR (n = 30). Consistent with the in vitro results, complement fragments C4a and Ba were increased in plasma from patients with AbMR compared to control subjects (P < 0.001 and P < 0.01, respectively). Endothelial microvesicle counts were not increased in patients with AbMR, however, and the number of microvesicles with C4 and C3 bound to the surface was actually lower compared to control subjects (both P < 0.05). Our results suggest that plasma complement activation fragments may be useful as non-invasive biomarkers of antibody-mediated complement activation within the allograft. Complement-opsonized endothelial microvesicles are decreased in patients with AbMR, possibly due to enhanced clearance of microvesicles opsonized with C3 and C4 fragments.

Biomarkers of Vascular Injury and Type 2 Diabetes: A Prospective Study, Systematic Review and Meta-Analysis.

Data on biomarkers of vascular injury and type 2 diabetes (T2D) risk from prospective studies are lacking. We evaluated seven biomarkers of vascular injury in relation to T2D. Additionally, a meta-analysis was performed. From the EPIC–Heidelberg cohort, 2224 participants were followed-up from baseline for 16 (median) years. E-Selectin, P-Selectin, intercellular adhesion molecule 3 (ICAM3), thrombomodulin, thrombopoietin, glycoprotein IIb/IIIa and fibrinogen levels were measured in baseline blood samples. The systematic review and meta-analysis included prospective studies identified through MEDLINE and Web of Science that investigated the association between mentioned biomarkers and T2D. The study population included 55% women, median age was 50 years, and 163 developed T2D. ICAM3 was associated with lower T2D risk (fully adjusted HRhighest vs. lowest tertile 0.62 (95% CI: 0.43, 0.91)), but no other studies on ICAM3 were identified. Overall, fifteen studies were included in the systematic review and meta-analysis (6,171 cases). E-Selectin was associated with higher T2D risk HRper SD: 1.34 (95% CI: 1.16, 1.54; I2 = 63%, n = 9 studies), while thrombomodulin was associated with lower risk HRper SD: 0.82 (95% CI: 0.71, 0.95; I2 = 0%, n = 2 studies). In the EPIC–Heidelberg, ICAM3 was associated with lower T2D risk. The meta-analysis showed a consistent positive association between E-Selectin and T2D. It was also suggestive of an inverse association between thrombomodulin and T2D, although further studies are needed to corroborate this finding.

A Pilot Study on Biomarkers of Vascular Injury in Patients with Immune-Mediated Thrombotic Thrombocytopenic Purpura

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is defined by thrombocytopenia and microangiopathic hemolytic anemia without an alternative explanation, confirmed by severely deficient ADAMTS13 to &lt;10%. It is caused by autoantibodies against the ADAMTS13 protease. Following recovery from an acute iTTP episode, the patient is at risk for relapses and multiple long-term complications including hypertension, depression, headaches and neurocognitive impairment. The etiology of these complications is not well understood. The aim of this study was to identify biomarkers of vascular injury that could help to diagnose or predict the development of these long term complications of iTTP. Four plasma biomarkers of vascular injury were selected with the assistance of a multidisciplinary team of neurologists, pathologists and psychologists, based on their relevance in other chronic diseases and inflammation. Syndecan-1 (CD-138), is a cell surface heparan sulfate proteoglycan that interacts with extracellular matrix molecules and growth factors to maintain epithelial cell morphology. It has been reported to be a negative regulator of various inflammatory processes, with Syndecan-1 knockout (Sdc-1−/−) mice showing enhanced disease severity and impaired recovery.Thrombomodulin (CD141), is an endothelial surface transmembrane glycoprotein. It is involved in the activation of protein C in the inactivation of thrombin. Its expression has been associated with aging and cardiovascular disease.Vascular adhesion protein-1 (VAP) is a member of the copper-containing amine oxidase/semicarbazide-sensitive amine oxidase (AOC/SSAO) enzyme family. It is continuously expressed as a transmembrane glycoprotein in the vascular wall during development and facilitates the accumulation of inflammatory cells into the inflamed environment. It has been shown to be released in cerebral ischemia.E-selectin (CD62E), is a selectin cell adhesion molecule, expressed only in endothelial cells activated by cytokines, and is known to be increased in acute coronary syndrome and atherosclerosis. Methods: Patients with a diagnosis of iTTP functioning normally in their activities of daily living were recruited from existing IRB approved patient cohorts at both the Ohio State University (n = 10) (Columbus) and the University College London Hospitals (n = 15) (London, UK). These patients were studied and reported previously (Cataland, Scully M et al. AJH 2011, 86; 87-89). with a validated measure of neurocognitive function (Cogstate). Patients were characterized by this testing as having normal or abnormal cognitive function. Syndecan-1, thrombomodulin, vascular adhesion protein-1, and E-selectin were studied in plasma samples obtained at the time of the previous neurocognitive testing. All four biomarker assays were performed with commercially available Elisa-based assays. Results: There was a total of 25 patients that underwent neurocognitive testing previously; 11 patients had normal neurocognitive testing and 14 patients were characterized as abnormal. The median age was 41 (range 18-62). One patient was Asian, six were black and 18 were white. Nine patients were male. The demographic information for each group is listed in Table 1. The biomarker results for each group are listed on Table 2 and a comparison is seen on Figure 1. We did see a trend to lower levels of Syndecan-1, and higher levels of thrombomodulin and VAP-1 in patients with abnormal cognitive testing. The two groups had similar levels of CD62E. all biomarker testing was repeated twice and given consistent data. Conclusions: There is a need in the field for a better understanding of the pathophysiology of the long-term complications described in patients with iTTP. This pilot study is the first to look at biomarkers of vascular injury and inflammation that can correlate with neurocognitive impairment and potentially vascular injury in patients with iTTP, not when patients are having an acute episode but during follow up. In addition, we have shown that these biomarkers can be reliably tested in frozen samples from patients with iTPP, and important step for feasibility of future studies. These data are an important first step to develop specific biomarkers that correlate with the development of long-term complications in iTTP patients. Disclosures Masias: Rigel Pharmaceuticals: Consultancy. Scully:Alexion: Consultancy; Novartis: Consultancy; Ablynx/Sanofi: Consultancy; Shire/Takeda: Consultancy; Shire: Research Funding. Cataland:Ablynx/Sanofi: Consultancy, Research Funding; Alexion: Consultancy, Research Funding.

Insulin protects against type 1 diabetes mellitus-induced aortopathy associated with the inhibition of biomarkers of vascular injury in rats

Background We sought to investigate the protective effect of insulin against type 1 diabetes mellitus (T1DM)-induced aortic injury (aortopathy) associated with the inhibition of biomarkers of vascular injury. Material and methods T1DM was induced in rats by streptozotocin (STZ) (65 mg/kg), and the protection group started insulin treatment 2 days post diabetic induction and continued until being sacrificed at week 8. Results Aortopathy was developed in the diabetic rats as demonstrated by profound alterations to the aorta ultrastructure, which was substantially protected by insulin. In addition, insulin significantly inhibited diabetes-induced dyslipidaemia, soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1), oxidative stress, and inflammation. However, blood levels of these biomarkers in the insulin-treated group were still significant (p < .05) compared with the control group, whereas insulin treatment returned blood glucose and triglyceride to control levels. Conclusions We demonstrate effective protection by insulin against T1DM-induced aortopathy in rats, which is associated with the inhibition of vascular injury biomarkers.