Assessment of the alpha 7 nicotinic acetylcholine receptor as an imaging marker of cardiac repair-associated processes using NS14490.

BACKGROUND: Cardiac repair and remodeling following myocardial infarction (MI) is a multifactorial process involving pro-reparative inflammation, angiogenesis and fibrosis. Noninvasive imaging using a radiotracer targeting these processes could be used to elucidate cardiac wound healing mechanisms. The alpha7 nicotinic acetylcholine receptor (ɑ7nAChR) stimulates pro-reparative macrophage activity and angiogenesis, making it a potential imaging biomarker in this context. We investigated this by assessing in vitro cellular expression of ɑ7nAChR, and by using a tritiated version of the PET radiotracer [18F]NS14490 in tissue autoradiography studies. RESULTS: ɑ7nAChR expression in human monocyte-derived macrophages and vascular cells showed the highest relative expression was within macrophages, but only endothelial cells exhibited a proliferation and hypoxia-driven increase in expression. Using a mouse model of inflammatory angiogenesis following sponge implantation, specific binding of [3H]NS14490 increased from 3.6 ± 0.2 µCi/g at day 3 post-implantation to 4.9 ± 0.2 µCi/g at day 7 (n = 4, P < 0.01), followed by a reduction at days 14 and 21. This peak matched the onset of vessel formation, macrophage infiltration and sponge fibrovascular encapsulation. In a rat MI model, specific binding of [3H]NS14490 was low in sham and remote MI myocardium. Specific binding within the infarct increased from day 14 post-MI (33.8 ± 14.1 µCi/g, P ≤ 0.01 versus sham), peaking at day 28 (48.9 ± 5.1 µCi/g, P ≤ 0.0001 versus sham). Histological and proteomic profiling of ɑ7nAChR positive tissue revealed strong associations between ɑ7nAChR and extracellular matrix deposition, and rat cardiac fibroblasts expressed ɑ7nAChR protein under normoxic and hypoxic conditions. CONCLUSION: ɑ7nAChR is highly expressed in human macrophages and showed proliferation and hypoxia-driven expression in human endothelial cells. While NS14490 imaging displays a pattern that coincides with vessel formation, macrophage infiltration and fibrovascular encapsulation in the sponge model, this is not the case in the MI model where the ɑ7nAChR imaging signal was strongly associated with extracellular matrix deposition which could be explained by ɑ7nAChR expression in fibroblasts. Overall, these findings support the involvement of ɑ7nAChR across several processes central to cardiac repair, with fibrosis most closely associated with ɑ7nAChR following MI.

Autophagy protein 5 controls flow-dependent endothelial functions.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

11ß-HSD1 inhibition does not affect murine tumour angiogenesis but may exert a selective effect on tumour growth by modulating inflammation and fibrosis.

Glucocorticoids inhibit angiogenesis by activating the glucocorticoid receptor. Inhibition of the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) reduces tissue-specific glucocorticoid action and promotes angiogenesis in murine models of myocardial infarction. Angiogenesis is important in the growth of some solid tumours. This study used murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC) to test the hypothesis that 11ß-HSD1 inhibition promotes angiogenesis and subsequent tumour growth. SCC or PDAC cells were injected into female FVB/N or C57BL6/J mice fed either standard diet, or diet containing the 11ß-HSD1 inhibitor UE2316. SCC tumours grew more rapidly in UE2316-treated mice, reaching a larger (P<0.01) final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). However, PDAC tumour growth was unaffected. Immunofluorescent analysis of SCC tumours did not show differences in vessel density (CD31/alpha-smooth muscle actin) or cell proliferation (Ki67) after 11ß-HSD1 inhibition, and immunohistochemistry of SCC tumours did not show changes in inflammatory cell (CD3- or F4/80-positive) infiltration. In culture, the growth/viability (assessed by live cell imaging) of SCC cells was not affected by UE2316 or corticosterone. Second Harmonic Generation microscopy showed that UE2316 reduced Type I collagen (P<0.001), whilst RNA-sequencing revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated SCC tumours. 11ß-HSD1 inhibition increases SCC tumour growth, likely via suppression of inflammatory/immune cell signalling and extracellular matrix deposition, but does not promote tumour angiogenesis or growth of all solid tumours.

Human embryonic stem cell-derived endothelial cell product injection attenuates cardiac remodeling in myocardial infarction.

Background: Mechanisms contributing to tissue remodeling of the infarcted heart following cell-based therapy remain elusive. While cell-based interventions have the potential to influence the cardiac healing process, there is little direct evidence of preservation of functional myocardium. Aim: The aim of the study was to investigate tissue remodeling in the infarcted heart following human embryonic stem cell-derived endothelial cell product (hESC-ECP) therapy. Materials and methods: Following coronary artery ligation (CAL) to induce cardiac ischemia, we investigated infarct size at 1 day post-injection in media-injected controls (CALM, n = 11), hESC-ECP-injected mice (CALC, n = 10), and dead hESC-ECP-injected mice (CALD, n = 6); echocardiography-based functional outcomes 14 days post-injection in experimental (CALM, n = 13; CALC, n = 17) and SHAM surgical mice (n = 4); and mature infarct size (CALM and CALC, both n = 6). We investigated ligand-receptor interactions (LRIs) in hESC-ECP cell populations, incorporating a publicly available C57BL/6J mouse cardiomyocyte-free scRNAseq dataset with naive, 1 day, and 3 days post-CAL hearts. Results: Human embryonic stem cell-derived endothelial cell product injection reduces the infarct area (CALM: 54.5 ± 5.0%, CALC: 21.3 ± 4.9%), and end-diastolic (CALM: 87.8 ± 8.9 uL, CALC: 63.3 ± 2.7 uL) and end-systolic ventricular volume (CALM: 56.4 ± 9.3 uL, CALC: 33.7 ± 2.6 uL). LRI analyses indicate an alternative immunomodulatory effect mediated via viable hESC-ECP-resident signaling. Conclusion: Delivery of the live hESC-ECP following CAL modulates the wound healing response during acute pathological remodeling, reducing infarct area, and preserving functional myocardium in this relatively acute model. Potential intrinsic myocardial cellular/hESC-ECP interactions indicate that discreet immunomodulation could provide novel therapeutic avenues to improve cardiac outcomes following myocardial infarction.

Comparison of mechanisms of angiostasis caused by the anti-inflammatory steroid 5α-tetrahydrocorticosterone versus conventional glucocorticoids.

5α-Tetrahydrocorticosterone (5αTHB) is an effective topical anti-inflammatory agent in mouse, with less propensity to cause skin thinning and impede new blood vessel growth compared with corticosterone. Its anti-inflammatory effects were not prevented by RU38486, a glucocorticoid receptor antagonist, suggesting alternative mechanisms. The hypothesis that 5αTHB directly inhibits angiogenesis to a lesser extent than hydrocortisone was tested, focussing on glucocorticoid receptor mediated actions. New vessel growth from aortae from C57BL/6 male mice was monitored in culture, in the presence of 5αTHB, hydrocortisone (mixed glucocorticoid/mineralocorticoid receptor agonist) or the selective glucocorticoid receptor agonist dexamethasone. Transcript profiles were studied, as was the role of the glucocorticoid receptor, using the antagonist, RU38486. Ex vivo, 5αTHB suppressed vessel growth from aortic rings, but was less potent than hydrocortisone (EC50 2512 nM 5αTHB, versus 762 nM hydrocortisone). In contrast to conventional glucocorticoids, 5αTHB did not alter expression of genes related to extracellular matrix integrity or inflammatory signalling, but caused a small increase in Per1 transcript, and decreased transcript abundance of Pecam1 gene. RU38486 did not antagonise the residual effects of 5αTHB to suppress vessel growth or regulate gene expression, but modified effects of dexamethasone. 5αTHB did not alter expression of glucocorticoid-regulated genes Fkbp51 and Hsd11b1, unlike hydrocortisone and dexamethasone. In conclusion, compared with hydrocortisone, 5αTHB exhibits limited suppression of angiogenesis, at least directly in blood vessels and probably independent of the glucocorticoid receptor. Discriminating the mechanisms employed by 5αTHB may provide the basis for the development of novel safer anti-inflammatory drugs for topical use.

Single-cell RNA sequencing profiling of mouse endothelial cells in response to pulmonary arterial hypertension.

AIMS: Endothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution. METHODS AND RESULTS: We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature. CONCLUSION: This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

The acute pressure natriuresis response is suppressed by selective ETA receptor blockade.

Hypertension is a major risk factor for cardiovascular disease.  In a significant minority of people, it develops when salt intake is increased (salt-sensitivity).  It is not clear whether this represents impaired vascular function or disruption to the relationship between blood pressure (BP) and renal salt-handling (pressure natriuresis, PN).  Endothelin-1 (ET-1) regulates BP via ETA and ETB receptor subtypes.  Blockade of ETA receptors reduces BP, but promotes sodium retention by an unknown mechanism.  ETB blockade increases both BP and sodium retention.  We hypothesised that ETA blockade promotes sodium and water retention by suppressing PN.  We also investigated whether suppression of PN might reflect off-target ETB blockade.  Acute PN was induced by sequential arterial ligation in male Sprague Dawley rats.  Intravenous atrasentan (ETA antagonist, 5mg/kg) halved the normal increase in medullary perfusion and reduced sodium and water excretion by >60%.  This was not due to off-target ETB blockade because intravenous A-192621 (ETB antagonist, 10mg/kg) increased natriuresis by 50% without modifying medullary perfusion.  In a separate experiment in salt-loaded rats monitored by radiotelemetry, oral atrasentan reduced systolic and diastolic BP by ~10mmHg, but additional oral A-192621 reversed these effects.  Endogenous ETA stimulation has natriuretic effects mediated by renal vascular dilation while endogenous ETB stimulation in the kidney has antinatriuretic effects via renal tubular mechanisms.  Pharmacological manipulation of vascular function with ET antagonists modifies the BP set-point, but even highly selective ETA antagonists attenuate PN, which may be associated with salt and water retention.

Positron Emission Tomography Techniques to Measure Active Inflammation, Fibrosis and Angiogenesis: Potential for Non-invasive Imaging of Hypertensive Heart Failure.

Heart failure, which is responsible for a high number of deaths worldwide, can develop due to chronic hypertension. Heart failure can involve and progress through several different pathways, including: fibrosis, inflammation, and angiogenesis. Early and specific detection of changes in the myocardium during the transition to heart failure can be made via the use of molecular imaging techniques, including positron emission tomography (PET). Traditional cardiovascular PET techniques, such as myocardial perfusion imaging and sympathetic innervation imaging, have been established at the clinical level but are often lacking in pathway and target specificity that is important for assessment of heart failure. Therefore, there is a need to identify new PET imaging markers of inflammation, fibrosis and angiogenesis that could aid diagnosis, staging and treatment of hypertensive heart failure. This review will provide an overview of key mechanisms underlying hypertensive heart failure and will present the latest developments in PET probes for detection of cardiovascular inflammation, fibrosis and angiogenesis. Currently, selective PET probes for detection of angiogenesis remain elusive but promising PET probes for specific targeting of inflammation and fibrosis are rapidly progressing into clinical use.

Human AdV-20-42-42, a Promising Novel Adenoviral Vector for Gene Therapy and Vaccine Product Development.

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

Glucocorticoids: Fuelling the Fire of Atherosclerosis or Therapeutic Extinguishers?

Glucocorticoids are steroid hormones with key roles in the regulation of many physiological systems including energy homeostasis and immunity. However, chronic glucocorticoid excess, highlighted in Cushing's syndrome, is established as being associated with increased cardiovascular disease (CVD) risk. Atherosclerosis is the major cause of CVD, leading to complications including coronary artery disease, myocardial infarction and heart failure. While the associations between glucocorticoid excess and increased prevalence of these complications are well established, the mechanisms underlying the role of glucocorticoids in development of atheroma are unclear. This review aims to better understand the importance of glucocorticoids in atherosclerosis and to dissect their cell-specific effects on key processes (e.g., contractility, remodelling and lesion development). Clinical and pre-clinical studies have shown both athero-protective and pro-atherogenic responses to glucocorticoids, effects dependent upon their multifactorial actions. Evidence indicates regulation of glucocorticoid bioavailability at the vasculature is complex, with local delivery, pre-receptor metabolism, and receptor expression contributing to responses linked to vascular remodelling and inflammation. Further investigations are required to clarify the mechanisms through which endogenous, local glucocorticoid action and systemic glucocorticoid treatment promote/inhibit atherosclerosis. This will provide greater insights into the potential benefit of glucocorticoid targeted approaches in the treatment of cardiovascular disease.

Regulatory Role of Sex Hormones in Cardiovascular Calcification.

Sex differences in cardiovascular disease (CVD), including aortic stenosis, atherosclerosis and cardiovascular calcification, are well documented. High levels of testosterone, the primary male sex hormone, are associated with increased risk of cardiovascular calcification, whilst estrogen, the primary female sex hormone, is considered cardioprotective. Current understanding of sexual dimorphism in cardiovascular calcification is still very limited. This review assesses the evidence that the actions of sex hormones influence the development of cardiovascular calcification. We address the current question of whether sex hormones could play a role in the sexual dimorphism seen in cardiovascular calcification, by discussing potential mechanisms of actions of sex hormones and evidence in pre-clinical research. More advanced investigations and understanding of sex hormones in calcification could provide a better translational outcome for those suffering with cardiovascular calcification.

MIR503HG Loss Promotes Endothelial-to-Mesenchymal Transition in Vascular Disease.

[Figure: see text].

CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice.

[Figure: see text].

Generation of a Novel In Vitro Model to Study Endothelial Dysfunction from Atherothrombotic Specimens.

PURPOSE: Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction. METHODS: In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo. RESULTS: CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo. CONCLUSIONS: CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.

Enhanced Angiogenesis by 11ßHSD1 Blockage Is Insufficient to Improve Reperfusion Following Hindlimb Ischaemia.

Background: Critical limb ischaemia (CLI), which is estimated to affect 2 million people in the United States, reduces quality of life, is associated with high morbidity and mortality, and has limited treatment options. Direct stimulation of angiogenesis using proangiogenic growth factors has been investigated as a therapeutic strategy to improve reperfusion in the ischaemic leg. Despite positive outcomes in animal studies, there has been little success in clinical translation. This investigation addressed the hypothesis that angiogenesis could be stimulated indirectly in the ischaemic hindlimb by blocking 11ß-hydroxysteroid dehydrogenase 1 (11ßHSD1)-mediated reactivation of anti-angiogenic glucocorticoids. Method and Results: Corticosterone suppressed ex vivo angiogenesis in the mouse aortic ring assay. 11ßHSD1 deletion (Hsd11b1Del1/Del1) or pharmacological inhibition (with 300 nM UE2316) which block the reactivation of glucocorticoid (i.e., the conversion of 11-dehydrocorticosterone (11DHC) to bioactive corticosterone) significantly reduced 11DHC-induced suppression of angiogenesis. In a sponge implantation model, 11ßHSD1 deletion, but not pharmacological inhibition, enhanced inflammation-induced angiogenesis. By contrast, in the mouse hindlimb ischaemia model, post-ischaemic reperfusion and vascular density were not affected by either deletion or pharmacological inhibition of 11ßHSD1 in young or aged mice. 3D vascular imaging suggested that hind limb reperfusion in the 1st week following induction of ischaemia may be driven by the rapid expansion of collateral arteries rather than by angiogenesis. Conclusion: 11ßHSD1-mediated glucocorticoid reactivation suppressed angiogenesis ex vivo and in vivo. However, regulation of angiogenesis alone was insufficient to promote reperfusion in hindlimb ischaemia. Future investigation of post-ischaemic reperfusion should include other aspects of systemic vascular remodeling including arteriogenesis and collateral formation.

Endothelin-1 Mediates the Systemic and Renal Hemodynamic Effects of GPR81 Activation.

GPR81 (G-protein-coupled receptor 81) is highly expressed in adipocytes, and activation by the endogenous ligand lactate inhibits lipolysis. GPR81 is also expressed in the heart, liver, and kidney, but roles in nonadipose tissues are poorly defined. GPR81 agonists, developed to improve blood lipid profile, might also provide insights into GPR81 physiology. Here, we assessed the blood pressure and renal hemodynamic responses to the GPR81 agonist, AZ'5538. In male wild-type mice, intravenous AZ'5538 infusion caused a rapid and sustained increase in systolic and diastolic blood pressure. Renal artery blood flow, intrarenal tissue perfusion, and glomerular filtration rate were all significantly reduced. AZ'5538 had no effect on blood pressure or renal hemodynamics in Gpr81-/- mice. Gpr81 mRNA was expressed in renal artery vascular smooth muscle, in the afferent arteriole, in glomerular and medullary perivascular cells, and in pericyte-like cells isolated from kidney. Intravenous AZ'5538 increased plasma ET-1 (endothelin 1), and pretreatment with BQ123 (endothelin-A receptor antagonist) prevented the pressor effects of GPR81 activation, whereas BQ788 (endothelin-B receptor antagonist) did not. Renal ischemia-reperfusion injury, which increases renal extracellular lactate, increased the renal expression of genes encoding ET-1, KIM-1 (Kidney Injury Molecule 1), collagen type 1-α1, TNF-α (tumor necrosis factor-α), and F4/80 in wild-type mice but not in Gpr81-/- mice. In summary, activation of GPR81 in vascular smooth muscle and perivascular cells regulates renal hemodynamics, mediated by release of the potent vasoconstrictor ET-1. This suggests that lactate may be a paracrine regulator of renal blood flow, particularly relevant when extracellular lactate is high as occurs during ischemic renal disease.

Non-invasive in vivo imaging of acute thrombosis: development of a novel factor XIIIa radiotracer.

AIMS: Cardiovascular thrombosis is responsible a quarter of deaths annually worldwide. Current imaging methods for cardiovascular thrombosis focus on anatomical identification of thrombus but cannot determine thrombus age or activity. Molecular imaging techniques hold promise for identification and quantification of thrombosis in vivo. Our objective was to assess a novel optical and positron-emitting probe targeting Factor XIIIa (ENC2015) as biomarker of active thrombus formation. METHODS AND RESULTS: Optical and positron-emitting ENC2015 probes were assessed ex vivo using blood drawn from human volunteers and passed through perfusion chambers containing denuded porcine aorta as a model of arterial injury. Specificity of ENC2015 was established with co-infusion of a factor XIIIa inhibitor. In vivo18F-ENC2015 biodistribution, kinetics, radiometabolism, and thrombus binding were characterized in rats. Both Cy5 and fluorine-18 labelled ENC2015 rapidly and specifically bound to thrombi. Thrombus uptake was inhibited by a factor XIIIa inhibitor. 18F-ENC2015 remained unmetabolized over 8 h when incubated in ex vivo human blood. In vivo, 42% of parent radiotracer remained in blood 60 min post-administration. Biodistribution studies demonstrated rapid clearance from tissues with elimination via the urinary system. In vivo,18F-ENC2015 uptake was markedly increased in the thrombosed carotid artery compared to the contralateral patent artery (mean standard uptake value ratio of 2.40 vs. 0.74, P < 0.0001). CONCLUSION: ENC2015 rapidly and selectively binds to acute thrombus in both an ex vivo human translational model and an in vivo rodent model of arterial thrombosis. This probe holds promise for the non-invasive identification of thrombus formation in cardiovascular disease.

Human Adipose-derived Pericytes Display Steroidogenic Lineage Potential in Vitro and Influence Leydig Cell Regeneration in Vivo in Rats.

Exogenous androgen replacement is used to treat symptoms associated with low testosterone in males. However, adverse cardiovascular risk and negative fertility impacts impel development of alternative approaches to restore/maintain Leydig cell (LC) androgen production. Stem Leydig cell (SLC) transplantation shows promise in this regard however, practicality of SLC isolation/transplantation impede clinical translation. Multipotent human adipose-derived perivascular stem cells (hAd-PSCs) represent an attractive extragonadal stem cell source for regenerative therapies in the testis but their therapeutic potential in this context is unexplored. We asked whether hAd-PSCs could be converted into Leydig-like cells and determined their capacity to promote regeneration in LC-ablated rat testes. Exposure of hAd-PSCs to differentiation-inducing factors in vitro upregulated steroidogenic genes but did not fully induce LC differentiation. In vivo, no difference in LC-regeneration was noted between Sham and hAd-PSC-transplanted rats. Interestingly, Cyp17a1 expression increased in hAd-PSC-transplanted testes compared to intact vehicle controls and the luteinising hormone/testosterone ratio returned to Vehicle control levels which was not the case in EDS + Sham animals. Notably, hAd-PSCs were undetectable one-month after transplantation suggesting this effect is likely mediated via paracrine mechanisms during the initial stages of regeneration; either directly by interacting with regenerating LCs, or through indirect interactions with trophic macrophages.