Thrombospondin-1 Plays a Major Pathogenic Role in Experimental and Human Bronchopulmonary Dysplasia.

Rationale: Extremely preterm infants develop bronchopulmonary dysplasia (BPD), a chronic lung injury that lacks effective treatment. TSP-1 (thrombospondin-1) is an antiangiogenic protein that activates TGF-ß1 (transforming growth factor-ß1), a cytokine strongly linked to both experimental and human BPD. Objectives:1) To examine effects of inhibiting TSP-1-mediated TGF-ß1 activation (LSKL [leucine-serine-lysine-leucine]) in neonatal rats with bleomycin-induced lung injury; 2) to examine effects of a TSP-1 mimic (ABT-510) on lung morphology; and 3) to determine whether TSP-1 and related signaling peptides are increased in lungs of human preterm infants at risk for BPD. Methods: From Postnatal Days 1 to 14, rat pups received daily intraperitoneal bleomycin (1 mg/kg) or vehicle and were treated with daily subcutaneous LSKL (20 mg/kg) or vehicle alone. Separate animals were treated with vehicle or ABT-510 (30 mg/kg/d). Paraffin-embedded lung tissues from 47 autopsies (controls; death <28 d, n = 30 and BPD at risk; death ⩾28 d, n = 17) performed on infants born <29 completed weeks' gestation were semiquantified for injury markers (collagen, macrophages, and 3-nitrotyrosine), TSP-1, and TGF-ß1. Measurements and Main Results: Bleomycin or ABT-510 increased lung TGF-ß1 activity and macrophage influx, caused pulmonary hypertension, and led to alveolar and microvascular hypoplasia. Treatment with LSKL partially prevented abnormal lung morphology secondary to bleomycin. Lungs from human infants at risk for BPD had increased contents of TSP-1 and TGF-ß1 when compared with controls. TGF-ß1 content correlated with markers of lung injury. Conclusions: TSP-1 inhibits alveologenesis in neonatal rats, in part via the upregulated activity of TGF-ß1. Observations in human lungs suggest a similar pathogenic role for TSP-1 in infants at risk for BPD.

Performance of Plasma Adenosine as a Biomarker for Predicting Cardiovascular Risk.

Adenosine boasts promising preclinical and clinical data supporting a vital role in modulating vascular homeostasis. Its widespread use as a diagnostic and therapeutic agent have been limited by its short half-life and complex biology, though adenosine-modulators have shown promise in improving vascular healing. Moreover, circulating adenosine has shown promise in predicting cardiovascular (CV) events. We sought to delineate whether circulating plasma adenosine levels predict CV events in patients undergoing invasive assessment for coronary artery disease. Patients undergoing invasive angiography had clinical data prospectively recorded in the Cardiovascular and Percutaneous ClInical TriALs (CAPITAL) revascularization registry and blood samples collected in the CAPITAL Biobank from which adenosine levels were quantified. Tertile-based analysis was used to assess prediction of major adverse cardiovascular events (MACE; composite of death, myocardial infarction, unplanned revascularization, and cerebrovascular accident). Secondary analyses included MACE subgroups, clinical subgroups and adenosine levels. There were 1,815 patients undergoing angiography who had blood collected with adenosine quantified in 1,323. Of those quantified, 51.0% were revascularized and 7.3% experienced MACE in 12 months of follow-up. Tertile-based analysis failed to demonstrate any stratification of MACE rates (log rank, P = 0.83), when comparing low-to-middle (hazard ratio (HR) 1.10, 95% confidence interval (CI) 0.68-1.78, P = 0.70) or low-to-high adenosine tertiles (HR 0.95, 95% CI 0.56-1.57, P = 0.84). In adjusted analysis, adenosine similarly failed to predict MACE. Finally, adenosine did not predict outcomes in patients with acute coronary syndrome nor in those revascularized or treated medically. Plasma adenosine levels do not predict subsequent CV outcomes or aid in patient risk stratification.

Evaluation of Plasma Adenosine as a Marker of Cardiovascular Risk: Analytical and Biological Considerations.

Background Adenosine is a ubiquitous regulatory molecule known to modulate signaling in many cells and processes vital to vascular homeostasis. While studies of adenosine receptors have dominated research in the field, quantification of adenosine systemically and locally remains limited owing largely to technical restrictions. Given the potential clinical implications of adenosine biology, there is a need for adequately powered studies examining the role of plasma adenosine in vascular health. We sought to describe the analytical and biological factors that affect quantification of adenosine in humans in a large, real-world cohort of patients undergoing evaluation for coronary artery disease. Methods and Results Between November 2016 and April 2018, we assessed 1141 patients undergoing angiography for evaluation of coronary artery disease. High-performance liquid chromatography was used for quantification of plasma adenosine concentration, yielding an analytical coefficient of variance (CVa) of 3.2%, intra-subject variance (CVi) 35.8% and inter-subject variance (CVg) 56.7%. Traditional cardiovascular risk factors, medications, and clinical presentation had no significant impact on adenosine levels. Conversely, increasing age (P=0.027) and the presence of obstructive coronary artery disease (P=0.026) were associated with lower adenosine levels. Adjusted multivariable analysis supported only age being inversely associated with adenosine levels (P=0.039). Conclusions Plasma adenosine is not significantly impacted by traditional cardiovascular risk factors; however, advancing age and presence of obstructive coronary artery disease may be associated with lower adenosine levels. The degree of intra- and inter-subject variance of adenosine has important implications for biomarker use as a prognosticator of cardiovascular outcomes and as an end point in clinical studies.

Development of an inflammation imaging tracer, 111In-DOTA-DAPTA, targeting chemokine receptor CCR5 and preliminary evaluation in an ApoE-/- atherosclerosis mouse model.

BACKGROUND: Chemokine receptor 5 (CCR5) plays an important role in atherosclerosis. Our objective was to develop a SPECT tracer targeting CCR5 for imaging plaque inflammation by radiolabeling D-Ala-peptide T-amide (DAPTA), a CCR5 antagonist, with 111In. METHODS: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated DAPTA (DOTA-DAPTA) was labeled with 111In. Cell uptake studies were conducted in U87-CD4-CCR5 and U87-MG cells. Biodistribution was determined in C57BL/6 mice. Autoradiography, en face and Oil Red O (ORO) imaging studies were performed in ApoE-/- mice. RESULTS: DOTA-DAPTA was radiolabeled with 111In with high radiochemical purity (> 98%) and specific activity (70 MBq·nmol). 111In-DOTA-DAPTA exhibited fast blood and renal clearance and high spleen uptake. The U87-CD4-CCR5 cells had significantly higher uptake in comparison to the U87-MG cells. The cell uptake was reduced by three times with DAPTA, indicating the receptor specificity of the uptake. Autoradiographic images showed significantly higher lesion uptake of 111In-DOTA-DAPTA in ApoE-/- mice than that in C57BL/6 mice. The tracer uptake in 4 month old ApoE-/- high fat diet (HFD) mice with blocking agent was twofold lower than the same mice without the blocking agent, demonstrating the specificity of the tracer for the CCR5 receptor. CONCLUSION: 111In-DOTA-DAPTA, specifically targeting chemokine receptor CCR5, is a potential SPECT agent for imaging inflammation in atherosclerosis.

Adenosine as a Marker and Mediator of Cardiovascular Homeostasis: A Translational Perspective.

Adenosine, a purine nucleoside, is produced broadly and implicated in the homeostasis of many cells and tissues. It signals predominantly via 4 purinergic adenosine receptors (ADORs) - ADORA1, ADORA2A, ADORA2B and ADOosine signaling, both through design as specific ADOR agonists and antagonists and as offtarget effects of existing anti-platelet medications. Despite this, adenosine has yet to be firmly established as either a therapeutic or a prognostic tool in clinical medicine to date. Herein, we provide a bench-to-bedside review of adenosine biology, highlighting the key considerations for further translational development of this proRA3 in addition to non-ADOR mediated effects. Through these signaling mechanisms, adenosine exerts effects on numerous cell types crucial to maintaining vascular homeostasis, especially following vascular injury. Both in vitro and in vivo models have provided considerable insights into adenosine signaling and identified targets for therapeutic intervention. Numerous pharmacologic agents have been developed that modulate adenmising molecule.

Role of plasminogen activator inhibitor-1 in coronary pathophysiology.

The standard of care for obstructive atherosclerotic coronary disease is revascularization, predominantly achieved via percutaneous placement of a stent with concurrent medical therapy. Advancements in percutaneous coronary intervention (PCI) have dramatically improved outcomes. However, major complications from PCI due to target lesion failure continue to occur at rates between 5 and 10% in the first twelve months following intervention limiting its therapeutic efficacy. Plasminogen activator inhibitor-1 (PAI-1) is a protein of interest for both arterial remodeling and thrombotic risk as it regulates cell migration and vascular thrombosis. Elevated PAI-1 antigen levels have been identified as a potential biomarker for coronary artery disease and metabolic syndrome while being modulated by a number of atherosclerotic risk factors. Although linked by some studies as a marker of disease severity and prognosis, it remains to be understood whether it is also a mediator and/or therapeutic target of vascular disease. In this review, we discuss the current understanding of PAI-1 in vascular disease and its potential role in in-stent restenosis and stent thrombosis.

Evaluation of Apoptosis with 99mTc-rhAnnexin V-128 and Inflammation with 18F-FDG in a Low-Dose Irradiation Model of Atherosclerosis in Apolipoprotein E-Deficient Mice.

Low-dose radiation in apolipoprotein E-deficient (ApoE-/-) mice has a protective effect with less subsequent atherosclerosis. Inflammation and apoptosis play major roles in the development of atherosclerosis. We evaluated the temporal pattern of the development of histologic atherosclerosis, inflammation with 18F-FDG, and apoptosis with 99mTc-rhAnnexin V-128 at 3 time points. METHODS: ApoE-/- mice were fed a high-fat diet, exposed to low-dose 60Co γ-radiation of 25 mGy at 2 mo of age, and evaluated within 1 wk (2-mo group), 1 mo (3-mo group), and 2 mo (4-mo group) from the time of radiation. Mice were divided into 3 subgroups and each received 18F-FDG, 99mTc-rhAnnexin V-128, or no radiotracer for autoradiography. Mice underwent euthanasia and aortic root dissection. The extent of atherosclerosis was determined by en face and Oil red O imaging. Aortic arch inflammation (18F-FDG) and apoptosis (99mTc-rhAnnexin V-128) were determined with digital autoradiography. Aortic sinus sections were stained with Sudan IV for assessment of lesion area and stage, antiCD68 antibody for inflammation and anti-cleaved-caspase 3 antibody for apoptosis. RESULTS: The extent of aortic atherosclerosis increased from 2 to 3 mo and from 3 to 4 mo. Inflammation (CD68) decreased and apoptosis (anti-cleaved-caspase 3 antibody) increased in aortic sinus slices measured as percentage of lesion by 4 mo. With increasing lesion stage, lesion inflammation decreased and lesion apoptosis increased. Aortic arch inflammation (18F-FDG uptake) did not differ over time and did not correlate with average lesion stage. However, aortic arch apoptosis (99mTc-rhAnnexin V-128) increased significantly by 4 mo and correlated with average lesion stage. There were no differences between the treatment subgroups (18F-FDG, 99mTc-rhAnnexin V-128, or no radiotracer). CONCLUSION: The temporal pattern of development of inflammation and apoptosis differ during the development of atherosclerosis in ApoE-/- mice treated with low-dose radiation. Advanced lesions are characterized by increased apoptosis and either less or similar amounts of inflammation, shown on immunohistochemistry and autoradiography. Treatment with radiotracers had no significant effects on extent of atherosclerosis, inflammation, or apoptosis.

Evidence that a synthetic amyloid-ß oligomer-binding peptide (ABP) targets amyloid-ß deposits in transgenic mouse brain and human Alzheimer's disease brain.

The synthetic ~5 kDa ABP (amyloid-ß binding peptide) consists of a region of the 228 kDa human pericentrioloar material-1 (PCM-1) protein that selectively and avidly binds in vitro Aß1-42 oligomers, believed to be key co-drivers of Alzheimer's disease (AD), but not monomers (Chakravarthy et al., (2013) [3]). ABP also prevents Aß1-42 from triggering the apoptotic death of cultured human SHSY5Y neuroblasts, likely by sequestering Aß oligomers, suggesting that it might be a potential AD therapeutic. Here we support this possibility by showing that ABP also recognizes and binds Aß1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aß1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice.

Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human Aß(1-42) and tau.

The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatin receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75(NTR)). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both Aß(1-42) and the mutant human tau protein tau(P301L,) the dentate granule cells still had immunostainable SSTR3- and p75(NTR)-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of Aß(1-42) or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of Aß(1-42) and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.

Involvement of insulin-like growth factor 1 receptor signaling in the amyloid-ß peptide oligomers-induced p75 neurotrophin receptor protein expression in mouse hippocampus.

The p75 neurotrophin receptor (p75NTR) has been thought to play a critical role in amyloid-ß peptide (Aß)-mediated neurodegeneration and Aß metabolism in Alzheimer's disease (AD) brains. Our previous report showed that membrane-associated p75NTR protein expression was significantly increased in the hippocampi of two different strains of transgenic AD mice and was associated with the age-dependent elevation of Aß1-42 levels. Here, we provide evidence that the Aß1-42 oligomers known as ADDLs (Aß-derived diffusible ligands) induce p75NTR protein expression through insulin-like growth factor 1 receptor (IGF-1R) phosphorylation in SH-SY5Y human neuroblastoma cells. An in vivo microinjection study demonstrated that microinjected ADDLs increased the p75NTR protein expression by 1.4-fold in the ipsilateral hippocampus compared to the contralateral hippocampus. In addition, ADDLs microinjected into mouse hippocampi facilitated IGF-1R phosphorylation within 30 min and the co-administration of picropodophyllin, an IGF-1R kinase inhibitor, blocked ADDLs-induced p75NTR expression. We examined the possible involvement of IGF-1R in the increased p75NTR protein expression in the hippocampi of 6-month-old AßPPswe/PS1dE9 AD model mice that had accumulated significant amounts of Aß1-42 and showed significantly higher p75NTR expression than age-matched wild-type mice. We found that IGF-1R phosphorylation in these transgenic mice was higher than that in the wild-type mice. These findings indicate that Aß1-42 oligomers stimulate the p75NTR protein expression in the hippocampus through IGF-1R signaling. Thus, Aß1-42 oligomers-mediated IGF-1R activation may trigger an increase in p75NTR protein expression in the hippocampus of AD brain during the early stages of disease development.

Hippocampal membrane-associated p75NTR levels are increased in Alzheimer's disease.

The pan-specific p75 neurotrophin receptor (p75(NTR)) is believed to play an important role in the pathogenesis of Alzheimer's disease (AD). It is involved in mediating amyloid-ß (Aß) toxicity and stimulating amyloidogenesis. In addition, we have recently shown that stimulating cultured SH-SY5Y human neuroblastoma cells with Aß(42) increases the level of membrane-associated p75(NTR) and that Aß(42)-accumation in two strains of transgenic AD model mice is accompanied by an increased level of hippocampal membrane-associated p75(NTR) (Chakravarthy et al. J Alzheimers Dis 19, 915-925, 2010). This raised an important question whether accumulating Aß(42) in human AD is also accompanied by an increased hippocampal membrane-associated p75(NTR). In this study, using polyclonal and monoclonal antibodies against the p75(NTR) receptor's intra- and extracellular domains, we show that indeed the mean level of membrane-associated p75(NTR) in the hippocampal formation is significantly higher (~two-fold, p < 0.03) in human AD brains than in identical samples of hippocampal formation in age-matched non-AD human brains. The possible relation of this elevated hippocampal p75(NTR) to AD cognitive decline is discussed.

The p75 neurotrophin receptor is localized to primary cilia in adult murine hippocampal dentate gyrus granule cells.

The densely ciliated granule cell layer of the adult murine hippocampal dentate gyrus is one of two sites of adult neurogenesis. The granule cells have already been proven to localize their SSTR3 (somatostatin receptor 3) receptors to their so-called primary cilia. Here we show for the first time that 70-90% of these cells in 7-18 months-old wild-type and 3×Tg-AD (Alzheimer disease transgenic) mice also load p75(NTR) receptors into the structures containing SSTR3, i.e., their primary cilia. On the other hand, p75(NTR')s TrkA co-receptors were not localized to cilia but conventionally distributed throughout the cell surface. Significantly fewer cells (20-40%) in the hippocampal CA1 and CA3 regions and cerebral cortex have p75(NTR) containing cilia. While we don't know what the impact of the cilial localization of p75(NTR) on dentate gyral adult neurogenesis and memory encoding might be, the cilia's amyloid ß-activatable p75(NTR) receptors could be damaging or lethal to the hippocampal functioning of amyloid ß-accumulating Alzheimer brain.

Amyloid-beta peptides stimulate the expression of the p75(NTR) neurotrophin receptor in SHSY5Y human neuroblastoma cells and AD transgenic mice.

The progression toward end-stage Alzheimer's disease (AD) in the aging brain is driven by accumulating amyloid-beta (Abeta)(1-42) oligomers that is accompanied by the downregulation of the Trk A neurotrophin receptor and by either upregulation or at least maintenance of the p75 neurotrophin receptor (p75(NTR)), which can be stimulated by the accumulating Abeta(1-42) peptides. Here we show that Abeta(1-42) and its active fragment Abeta(25-35), but not Abeta(42-1), can at least double the level of p75(NTR) receptors in the membranes of model SH-SY5Y human neuroblastoma cells. We also show that p75(NTR) is upregulated in the hippocampi of two strains of AD transgenic mice. Specifically, the level of the p75(NTR) receptor in the hippocampal membranes from 12-15 month old AD-triple transgenic mice (3xTg-AD) harboring PS1 (M146V), AbetaPP (Swe), and tau (P301L) was nearly twice that in hippocampal membranes from age-matched wild-type mice. Similarly, the level of p75 (NTR) receptor in 7 month-old B6.Cg-Tg AD mice harboring PSEN1dE9 and AbetaPP (Swe) was also increased above the level in the corresponding wild-type mice. This increase correlated with the age-dependent rise in Abeta(1-42) levels in the AD mice. Thus, it appears that it could be the accumulating Abeta(1-42) that increases or at least prevents the downregulation of p75 (NTR) receptors in key parts of AD brains. It is possible that when the Abeta (1-42) accumulation reaches a critical level in the brain on the way to late-onset AD, the Abeta (1-42) induced p75 (NTR) receptor signaling starts a vicious cycle that accelerates AD development because of the activated receptors' recently shown ability to stimulate Abeta(1-42) production.