Improved on-tissue detection of the anti-cancer agent doxorubicin by quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging.

Doxorubicin (dox) is an affordable, and highly effective chemotherapeutic agent used in cancer treatment, yet its application is known to cause cumulative cardiac and renal toxicity. In this study, we employed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to evaluate the distribution of dox in mouse heart and kidney after in vivo treatment. To this end, we performed absolute quantification using an isotopically labeled form (13C d3-dox) as an internal standard. Unfortunately, ion suppression often leads to loss of sensitivity in compound detection and can result in hampered drug quantification. To overcome this issue, we developed an on-tissue chemical derivatization (OTCD) method using Girard's reagent T (GirT). With the developed method, dox signal was increased by two orders of magnitude. This optimized sample preparation enabled a sensible gain in dox detection, making it possible to study its distribution and abundance (up to 0.11 pmol/mm2 in the heart and 0.33 pmol/mm2 in the kidney medulla). The optimized approach for on-tissue derivatization and subsequent quantification creates a powerful tool to better understand the relationship between dox exposure (at clinically relevant concentrations) and its biological detrimental effects in various tissues. Overall, this work is a showcase of the added value of MALDI-MSI for pharmaceutical studies to better understand heterogeneity in drug exposure between and within organs.

Doxorubicin Impairs Smooth Muscle Cell Contraction: Novel Insights in Vascular Toxicity.

Clinical and animal studies have demonstrated that chemotherapeutic doxorubicin (DOX) increases arterial stiffness, a predictor of cardiovascular risk. Despite consensus about DOX-impaired endothelium-dependent vasodilation as a contributing mechanism, some studies have reported conflicting results on vascular smooth muscle cell (VSMC) function after DOX treatment. The present study aimed to investigate the effects of DOX on VSMC function. To this end, mice received a single injection of 4 mg DOX/kg, or mouse aortic segments were treated ex vivo with 1 µM DOX, followed by vascular reactivity evaluation 16 h later. Phenylephrine (PE)-induced VSMC contraction was decreased after DOX treatment. DOX did not affect the transient PE contraction dependent on Ca2+ release from the sarcoplasmic reticulum (0 mM Ca2+), but it reduced the subsequent tonic phase characterised by Ca2+ influx. These findings were supported by similar angiotensin II and attenuated endothelin-1 contractions. The involvement of voltage-gated Ca2+ channels in DOX-decreased contraction was excluded by using levcromakalim and diltiazem in PE-induced contraction and corroborated by similar K+ and serotonin contractions. Despite the evaluation of multiple blockers of transient receptor potential channels, the exact mechanism for DOX-decreased VSMC contraction remains elusive. Surprisingly, DOX reduced ex vivo but not in vivo arterial stiffness, highlighting the importance of appropriate timing for evaluating arterial stiffness in DOX-treated patients.

Serum Corticosterone and Insulin Resistance as Early Biomarkers in the hAPP23 Overexpressing Mouse Model of Alzheimer's Disease.

Increasing epidemiological evidence highlights the association between systemic insulin resistance and Alzheimer's disease (AD). As insulin resistance can be caused by high-stress hormone levels and since hypercortisolism appears to be an important risk factor of AD, we aimed to investigate the systemic insulin functionality and circulating stress hormone levels in a mutant humanized amyloid precursor protein (APP) overexpressing (hAPP23+/-) AD mouse model. Memory and spatial learning of male hAPP23+/- and C57BL/6 (wild type, WT) mice were assessed by a Morris Water Maze (MWM) test at the age of 4 and 12 months. The systemic metabolism was examined by intraperitoneal glucose and insulin tolerance tests (GTT, ITT). Insulin and corticosterone levels were determined in serum. In the hippocampus, parietal and occipital cortex of hAPP23+/- brains, amyloid-beta (Aß) deposits were present at 12 months of age. MWM demonstrated a cognitive decline in hAPP23+/- mice at 12 but not at 4 months, evidenced by increasing total path lengths and deteriorating probe trials compared to WT mice. hAPP23+/- animals presented increased serum corticosterone levels compared to WT mice at both 4 and 12 months. hAPP23+/- mice exhibited peripheral insulin resistance compared to WT mice at 4 months, which stabilized at 12 months of age. Serum insulin levels were similar between genotypes at 4 months of age but were significantly higher in hAPP23+/- mice at 12 months of age. Peripheral glucose homeostasis remained unchanged. These results indicate that peripheral insulin resistance combined with elevated circulating stress hormone levels could be potential biomarkers of the pre-symptomatic phase of AD.

A systematic literature review on the effects of exercise on human Toll-like receptor expression.

BACKGROUND: Toll-like receptors (TLRs) are a family of transmembrane pattern recognition receptors that are mainly expressed on immune cells. Recognition of various exogenous and endogenous molecular patterns activates the TLR signalling cascade, which orchestrates an inflammatory immune response. Dysfunctional immune responses, including aberrant TLR signalling, are increasingly implicated in the associations between sedentarism, chronic low-grade systemic inflammation and various non-communicable diseases. Conversely, exercise exerts anti-inflammatory effects, which could be conferred through its immunomodulatory properties, potentially affecting TLRs. This study aims to systematically review the effects of exercise on human TLR expression. METHOD: A systematic literature search of Pubmed, Embase, The Cochrane Library and SPORTDiscus for articles addressing the impact of exercise (as isolated intervention) on TLRs in humans was conducted, ending in February 2020. RESULTS: A total of 66 articles were included. The publications were categorised according to exercise modality and duration: acute resistance exercise (4 studies), acute aerobic exercise (26 studies), resistance training program (9 studies), aerobic training program (16 studies), combined (i.e. resistance and aerobic) training program (8 studies) and chronic exercise not otherwise classifiable (9 studies). Five articles investigated more than one of the aforementioned exercise categories. Several trends could be discerned with regard to the TLR response in the different exercise categories. Acute resistance exercise seemed to elicit TLR upregulation, whereas acute aerobic exercise had less activating potential with the majority of responses being neutral or, especially in healthy participants, downregulatory. Chronic resistance and combined exercise programs predominantly resulted in unaltered or decreased TLR levels. In the chronic aerobic exercise category, mixed effects were observed, but the majority of measurements demonstrated unchanged TLR expression. CONCLUSION: Currently published research supports an interplay between exercise and TLR signalling, which seems to depend on the characteristics of the exercise. However, there was large heterogeneity in the study designs and methodologies. Therefore, additional research is required to further corroborate these findings, to define its pathophysiological implications and to elucidate the mechanism(s) linking exercise to TLR signalling.

Neuregulin-1 compensates for endothelial nitric oxide synthase deficiency.

Endothelial cells (ECs) secrete different paracrine signals that modulate the function of adjacent cells; two examples of these paracrine signals are nitric oxide (NO) and neuregulin-1 (NRG1), a cardioprotective growth factor. Currently, it is undetermined whether one paracrine factor can compensate for the loss of another. Herein, we hypothesized that NRG1 can compensate for endothelial NO synthase (eNOS) deficiency. We characterized eNOS null and wild-type (WT) mice by cardiac ultrasound and histology and we determined circulating NRG1 levels. In a separate experiment, eight groups of mice were divided into four groups of eNOS null mice and WT mice; half of the mice received angiotensin II (ANG II) to induce a more severe phenotype. Mice were randomized to daily injections with NRG1 or vehicle for 28 days. eNOS deficiency increased NRG1 plasma levels, indicating that ECs increase their NRG1 expression when NO production is deleted. eNOS deficiency also increased blood pressure, lowered heart rate, induced cardiac fibrosis, and affected diastolic function. In eNOS null mice, ANG II administration not only increased cardiac fibrosis but also induced cardiac hypertrophy and renal fibrosis. NRG1 administration prevented cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. Moreover, Nrg1 expression in the myocardium is shown to be regulated by miR-134. This study indicates that administration of endothelium-derived NRG1 can compensate for eNOS deficiency in the heart and kidneys.NEW & NOTEWORTHY ECs compensate for eNOS deficiency by increasing the secretion of NRG1. NRG1 administration prevents cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. NRG1 expression is regulated by miR-134.

Doxorubicin induces arterial stiffness: A comprehensive in vivo and ex vivo evaluation of vascular toxicity in mice.

Arterial stiffness is an important predictor of cardiovascular risk. Clinical studies have demonstrated that arterial stiffness increases in cancer patients treated with the chemotherapeutic doxorubicin (DOX). However, the mechanisms of DOX-induced arterial stiffness remain largely unknown. This study aimed to evaluate artery stiffening in DOX-treated mice using in vivo and ex vivo techniques. Male C57BL/6J mice were treated for 2 weeks with 2 mg/kg (low dose) or 4 mg/kg (high dose) of DOX weekly. Arterial stiffness was assessed in vivo with ultrasound imaging (abdominal aorta pulse wave velocity (aaPWV)) and applanation tonometry (carotid-femoral PWV) combined with ex vivo vascular stiffness and reactivity evaluation. The high dose increased aaPWV, while cfPWV did not reach statistical significance. Phenylephrine (PE)-contracted aortic segments showed a higher Peterson's modulus (Ep) in the high dose group, while Ep did not differ when vascular smooth muscle cells (VSMCs) were relaxed by a NO donor (DEANO). In addition, aortic rings of DOX-treated mice showed increased PE contraction, decreased basal nitric oxide (NO) index and impaired acetylcholine-induced endothelium-dependent relaxation. DOX treatment contributed to endothelial cell loss and reduced endothelial nitric oxide synthase (eNOS) expression in the aorta. In conclusion, we have replicated DOX-induced arterial stiffness in a murine model and this aortic stiffness is driven by impaired endothelial function, contributing to increased vascular tone.

INSPIRE: A European training network to foster research and training in cardiovascular safety pharmacology.

Safety pharmacology is an essential part of drug development aiming to identify, evaluate and investigate undesirable pharmacodynamic properties of a drug primarily prior to clinical trials. In particular, cardiovascular adverse drug reactions (ADR) have halted many drug development programs. Safety pharmacology has successfully implemented a screening strategy to detect cardiovascular liabilities, but there is room for further refinement. In this setting, we present the INSPIRE project, a European Training Network in safety pharmacology for Early Stage Researchers (ESRs), funded by the European Commission's H2020-MSCA-ITN programme. INSPIRE has recruited 15 ESR fellows that will conduct an individual PhD-research project for a period of 36 months. INSPIRE aims to be complementary to ongoing research initiatives. With this as a goal, an inventory of collaborative research initiatives in safety pharmacology was created and the ESR projects have been designed to be complementary to this roadmap. Overall, INSPIRE aims to improve cardiovascular safety evaluation, either by investigating technological innovations or by adding mechanistic insight in emerging safety concerns, as observed in the field of cardio-oncology. Finally, in addition to its hands-on research pillar, INSPIRE will organize a number of summer schools and workshops that will be open to the wider community as well. In summary, INSPIRE aims to foster both research and training in safety pharmacology and hopes to inspire the future generation of safety scientists.

Neuregulin-1 attenuates stress-induced vascular senescence.

Aims: Cardiovascular ageing is a key determinant of life expectancy. Cellular senescence, a state of irreversible cell cycle arrest, is an important contributor to ageing due to the accumulation of damaged cells. Targeting cellular senescence could prevent age-related cardiovascular diseases. In this study, we investigated the effects of neuregulin-1 (NRG-1), an epidermal growth factor with cardioprotective and anti-atherosclerotic effects, on cellular senescence. Methods and results: Senescence was induced in cultured rat aortic endothelial cells (ECs) and aortic smooth muscle cells (SMCs) by 2 h exposure to 30 µM hydrogen peroxide (H2O2). Cellular senescence was confirmed after 72 h using senescence-associated-ß-galactosidase staining (SA-ß-gal), cell surface area, and western blot analyses of SA pathways (acetyl-p53, p21). Recombinant human NRG-1 (rhNRG-1, 20 ng/mL) significantly reduced H2O2-induced senescence, as shown by a lower number of SA-ß-gal positive cells, smaller surface area and lower expression of acetyl-p53. In C57BL/6 male mice rendered diabetic with streptozotocin (STZ), rhNRG-1 attenuated cellular senescence in aortic ECs and SMCs. Next, we created mice with SMC-specific knockdown of the NRG-1 receptor ErbB4. Aortic SMCs isolated from SMC-specific ErbB4 deficient mice (ErbB4f/+ SM22α-Cre+) showed earlier cellular senescence in vitro compared with wild-type (ErbB4+/+ SM22α-Cre+) SMCs. Furthermore, when rendered diabetic with STZ, ErbB4f/+ SM22α-Cre+ male mice showed significantly more vascular senescence than their diabetic wild-type littermates and had increased mortality. Conclusions: This study is the first to explore the role of NRG-1 in vascular senescence. Our data demonstrate that NRG-1 markedly inhibits stress-induced premature senescence in vascular cells in vitro and in the aorta of diabetic mice in vivo. Consistently, deficiency in the NRG-1 receptor ErbB4 provokes cellular senescence in vitro as well as in vivo.

Attenuated atherogenesis in apolipoprotein E-deficient mice lacking amyloid precursor protein.

OBJECTIVE: Recent evidence suggests that amyloid precursor protein (APP) is overexpressed in atherosclerosis-prone regions of mouse aorta. We therefore investigated in the present study whether APP has a role in the progression and composition of atherosclerotic plaques. METHODS AND RESULTS: Apolipoprotein E-deficient (apoE(-/-)) mice were crossbred with animals lacking APP (APP(-/-)). After 16 weeks on a Western-type diet, apoE(-/-) and APP(-/-)/apoE(-/-) mice showed similar cholesterol levels. However, atherosclerotic plaque size was significantly reduced in the distal thoracic aorta (90% reduction) and abdominal aorta (75% reduction) of APP(-/-)/apoE(-/-) mice as compared to apoE(-/-). Plaques at the level of the aortic valves were not different in size, but showed a more stable phenotype in APP(-/-)/apoE(-/-) mice, as indicated by a reduced macrophage content, an increased amount of collagen and a thicker fibrous cap. CONCLUSION: Our findings provide evidence that lack of APP attenuates atherogenesis and leads to plaque stability.

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND AND PURPOSE: P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis. EXPERIMENTAL APPROACH: mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE(-/-)) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE(-/-) mice. KEY RESULTS: P2Y(6) receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y(2) receptor expression remained unchanged. Expression of P2Y(1) or P2Y(4) receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y(6) mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y(6)-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y(6) receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y(6)-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100-300 microM) or pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS, 10-30 microM). Finally, 4-week treatment of cholesterol-fed apoE(-/-) mice with suramin or PPADS (50 and 25 mg.kg(-1).day(-1) respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication. CONCLUSIONS AND IMPLICATIONS: These results suggest involvement of nucleotide receptors, particularly P2Y(6) receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y(6) receptor-deficient mice.

Endothelium-dependent relaxation evoked by ATP and UTP in the aorta of P2Y2-deficient mice.

Based on pharmacological criteria, we previously suggested that in the mouse aorta, endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (adenosine diphosphate (ADP)), P2Y2 (adenosine triphosphate (ATP)) and P2Y6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild-type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling. Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and adenosine 5'[gamma-thio] triphosphate (ATPgammaS)-evoked relaxation, suggesting that in WT mice, ATP and ATPgammaS activate predominantly the P2Y2 subtype. The ATP/ATPgammaS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, as they were sensitive to 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS2179), a P2Y1-selective antagonist. In contrast to ATP, the UTP-evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6(like) receptor subtype. In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6(like) receptor.

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta.

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5' triphosphate (ATP), uridine 5' triphosphate (UTP), uridine 5' diphosphate (UDP); >90%) or partial (adenosine 5' diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP approximately UTP approximately ADP>adenosine 5'-[gamma-thio] triphosphate (ATPgammaS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53+/-0.07) was compatible with literature, but the pKb for UTP (5.19+/-0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2'-deoxy-N6-methyladenosine3',5'-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.