Although the hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used in atherosclerosis to reduce serum cholesterol, statins have multiple other effects, including direct ...effects on cells of the vessel wall. Recently, DNA damage, including telomere shortening, has been identified in vascular smooth muscle cells (VSMCs) in human atherosclerosis. Although statins reduce DNA damage in vitro, the mechanisms by which they might protect DNA integrity in VSMCs are unknown. We show that human atherosclerotic plaque VSMCs exhibit increased levels of double-stranded DNA breaks and basal activation of DNA repair pathways involving ataxia telangiectasia–mutated (ATM) and the histone H2AX in vivo and in vitro. Oxidant stress induced DNA damage and activated DNA repair pathways in VSMCs. Statin treatment did not reduce oxidant stress or DNA damage but markedly accelerated DNA repair. Accelerated DNA repair required both the Nijmegen breakage syndrome (NBS)-1 protein and the human double minute protein Hdm2, accompanied by phosphorylation of Hdm2, dissociation of NBS-1 and Hdm2, inhibition of NBS-1 degradation, and accelerated phosphorylation of ATM. Statin treatment reduced VSMC senescence and telomere attrition in culture, accelerated DNA repair and reduced apoptosis in vivo after irradiation, and reduced ATM/ATR (ATM and Rad3-related) activity in atherosclerosis. We conclude that statins activate a novel mechanism of accelerating DNA repair, dependent on NBS-1 stabilization and Hdm2. Statin treatment may delay cell senescence and promote DNA repair in atherosclerosis.
Although human atherosclerosis is associated with aging, direct evidence of cellular senescence and the mechanism of senescence in vascular smooth muscle cells (VSMCs) in atherosclerotic plaques is ...lacking. We examined normal vessels and plaques by histochemistry, Southern blotting, and fluorescence in situ hybridization for telomere signals. VSMCs in fibrous caps expressed markers of senescence (senescence-associated β-galactosidase SAβG and the cyclin-dependent kinase inhibitors cdkis p16 and p21) not seen in normal vessels. In matched samples from the same individual, plaques demonstrated markedly shorter telomeres than normal vessels. Fibrous cap VSMCs exhibited markedly shorter telomeres compared with normal medial VSMCs. Telomere shortening was closely associated with increasing severity of atherosclerosis. In vitro, plaque VSMCs demonstrated morphological features of senescence, increased SAβG expression, reduced proliferation, and premature senescence. VSMC senescence was mediated by changes in cyclins D/E, p16, p21, and pRB, and plaque VSMCs could reenter the cell cycle by hyperphosphorylating pRB. Both plaque and normal VSMCs expressed low levels of telomerase. However, telomerase expression alone rescued plaque VSMC senescence despite short telomeres, normalizing the cdki/pRB changes. In vivo, plaque VSMCs exhibited oxidative DNA damage, suggesting that telomere damage may be induced by oxidant stress. Furthermore, oxidants induced premature senescence in vitro, with accelerated telomere shortening and reduced telomerase activity. We conclude that human atherosclerosis is characterized by senescence of VSMCs, accelerated by oxidative stress-induced DNA damage, inhibition of telomerase and marked telomere shortening. Prevention of cellular senescence may be a novel therapeutic target in atherosclerosis.
Aldosterone is released from adrenal zona glomerulosa (ZG) cells and plays an important role in Na and K homoeostasis. Mutations in the human inwardly rectifying K channel CNJ type (
)
(
) gene ...encoding the G-coupled inwardly rectifying K channel 4 (GIRK4) cause abnormal aldosterone secretion and hypertension. To better understand the role of wild-type (WT) GIRK4 in regulating aldosterone release, we have looked at aldosterone secretion in a
knockout (KO) mouse. We found that female but not male KO mice have reduced aldosterone levels compared with WT female controls, but higher levels of aldosterone after angiotensin II (Ang-II) stimulation. These differences could not be explained by sex differences in aldosterone synthase (
) gene expression in the mouse adrenal. Using RNAseq analysis to compare WT and KO adrenals, we showed that females also have a much larger set of differentially expressed adrenal genes than males (395 compared with 7). Ingenuity Pathway Analysis (IPA) of this gene set suggested that peroxisome proliferator activated receptor (PPAR) nuclear receptors regulated aldosterone production and altered signalling in the female KO mouse, which could explain the reduced aldosterone secretion. We tested this hypothesis in H295R adrenal cells and showed that the selective PPARα agonist fenofibrate can stimulate aldosterone production and induce Cyp11b2. Dosing mice
produced similar results. Together our data show that
is important for baseline aldosterone secretion, but its importance is sex-limited at least in the mouse. It also highlights a novel regulatory pathway for aldosterone secretion through PPARα that may have translational potential in human hyperaldosteronism.
Cell cycle inhibitors are promising agents to prevent or treat human coronary in-stent stenosis (ISS). However, their lack of specificity for ISS vascular smooth muscle cells (VSMCs) may inhibit ...medial VSMC proliferation and suppress vessel healing.
To identify inhibitor targets that differentially regulate proliferation of ISS vs. medial VSMCs, we examined cell cycle regulation in human VSMCs derived from (A) normal media, (B) ISS sites and (C) primary atherosclerotic plaques (P-VSMCs) using time-lapse videomicroscopy, flow cytometry, immunoblotting and immunohistochemistry.
ISS-VSMC proliferation was intermediate between P-VSMCs and medial VSMCs. Compared with medial cells, P-VSMCs expressed increased p16 and p21, reduced p27, reduced cyclins D(1) and E, and reduced pRb phosphorylation. In contrast, ISS-VSMCs expressed high levels of cyclins E and A with pRb hyperphosphorylation, both in vitro and in vivo, associated with increased and chronic cell proliferation in vivo. Roscovitine, a selective CDK2 inhibitor, inhibited VSMC proliferation by both pRb-dependent and independent pathways and more potently in ISS-VSMCs than medial VSMCs.
Human ISS-VSMCs have marked differences in the stable expression of multiple cell cycle regulators, suggesting that ISS-VSMCs derive from P-VSMCs driven to proliferate through cyclin E overexpression. The critical role for cyclin E-CDK2 enables the identification of the first agent that selectively inhibits ISS-VSMC proliferation.
BackgroundThe epicardium plays a pivotal role in embryonic heart development by providing trophic and structural support to the developing and adult myocardium.HypothesisWe hypothesized that human ...embryonic stem cell-derived (hESC) epicardium would augment the structure and function of engineered heart tissues (EHT) in vitro and of new myocardium in vivo following hESC-cardiomyocyte transplantation.MethodsHESC-epicardial cells were co-cultured with hESC-cardiomyocytes in 3D-EHTs for 14 days and compared to hESC-mesenchymal stromal cells (MSC), primary MSC or cardiomyocytes alone. We measured cardiomyocyte structural maturation along with Ca-handling and force generation. We next investigated the efficacy of hESC-epicardium to augment hESC-cardiomyocyte grafts in infarcted athymic rat hearts. Athymic rats underwent a 60-minute ischaemia-reperfusion injury, which after 4 days was followed by intramyocardial transplantation of either 5x10 epicardial cells, 10x10 cardiomyocytes, 5x10 epicardial cells plus 10x10 cardiomyocytes or vehicle control (n=60). Animals were subjected to functional assessment by cardiac ultrasound and post-mortem histological analysis. RNA-sequencing was performed to elucidate putative mediators in epicardial driven heart repair.ResultsEpicardial cells markedly enhanced the myofibril structure, calcium handling and force generation of human EHTs, while reducing passive stiffness compared to mesenchymal stromal cells. Grafted epicardial cells formed robust fibroblast grafts in the infarct zone up to 3 months post transplantation. Co-transplantation of hESC-epicardial cells and cardiomyocytes doubled graft cardiomyocyte proliferation rates in vivo, resulting in 2.5-fold greater cardiac graft size, simultaneously augmenting graft and host vascularization as well as graft maturation. Importantly, co-transplantation improved systolic function compared with hearts receiving either cardiomyocytes alone, epicardial cells alone or vehicle, and these functional benefits were maintained out to 3 months post-transplantation. Finally, RNA-sequencing revealed the epicardial secretome demonstrating abundant expression of fibronectin for which epicardial cells are required.ConclusionEpicardial cells promote cardiomyocyte structure and contractility in vitro and augment cardiac graft size and function in vivo. These cells therefore contribute to the formation of healthier new myocardium and are a promising adjuvant therapeutic for cardiac repair.
Interindividual variability in paclitaxel and docetaxel pharmacokinetics, toxicity and response is extensive, and largely unexplained. We hypothesized that this is due to affinity of taxanes for an ...uptake transporter that indirectly regulates elimination pathways. Here, we studied accumulation of 3Hdocetaxel and 3Hpaclitaxel in Xenopus laevis oocytes injected with cRNA of the liver-specific organic anion transporting polypeptide (OATP) family members OATP1B1 (OATP2) or OATP1B3 (OATP8). Taxane transport by OATP1B1 expressing oocytes was not significantly different from that by water-injected controls, whereas uptake by OATP1B3 was 2.2-fold higher for docetaxel (P=0.0007) and 3.3-fold higher for paclitaxel (P<0.0001). OATP1B3-mediated paclitaxel transport was saturable (Michaelis-Menten constant, 6.79 ?M), time-dependent, and highly sensitive to chemical inhibition. Paclitaxel uptake was not inhibited by ketoconazole or tariquidar. However, uptake was inhibited by the formulation excipient Cremophor (74.4% inhibition, P<0.0001), cyclosporin A (25.2%, P=0.005), glycyrrhizic acid (24.6%, P=0.012), and hyperforin (28.4%, P=0.003). Consistent with this finding, Cremophor was found to significantly affect the hepatic uptake of paclitaxel in mice. These data suggest that OATP1B3 is a key regulator of hepatic uptake, and may therefore play a role in the variable response to treatment with taxanes.
The anticancer agent irinotecan (CPT-11) is converted to SN-38, which is approximately 100 to 1000-fold more cytotoxic than the parent drug. The pharmacokinetics of irinotecan are extremely complex ...and have been the subject of intensive investigation in recent years. Irinotecan is subject to extensive metabolism by various polymorphic enzymes, including CES2 to form SN-38, members of the UGT1A subfamily, and CYP3A4 and CYP3A5, which form several pharmacologically inactive oxidation products. Elimination of irinotecan is also dependent on drug-transporting proteins, notably ABCB1 (P-glycoprotein), ABCC2 (cMOAT) and ABCG2 (BCRP), present on the bile canalicular membrane. The various processes mediating drug elimination, either through metabolic breakdown or excretion, likely impact substantially on interindividual variability in drug handling. This report provides an update on current strategies to individualize irinotecan chemotherapy based on each patient’s genetic constitution, which may ultimately lead to more selective use of this agent.
Despite its controversial past, thalidomide is currently under investigation for the treatment of several disease types, ranging from inflammatory conditions to cancer. The mechanism of action of ...thalidomide is complex and not yet fully understood, but there is some evidence to suggest that metabolism may play a role. Consequently, there has been a considerable effort to characterize the metabolism of thalidomide in recent years. Thalidomide undergoes biotransformation by non-enzymatic hydrolysis and enzyme-mediated hydroxylation to form a multitude of metabolites. Metabolite identification and reaction phenotyping studies have been performed and will be discussed in this review in addition to interspecies differences in thalidomide metabolism.
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