Giorgio Aquila

Mitochondrial permeability transition involves dissociation of F1FO ATP synthase dimers and C-ring conformation

The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F1FO ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F1FO ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F1FO ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT. Specific mutations in the F1FO ATP synthase c subunit that alter C‐ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F1FO ATP synthase dimers. Destabilizing F1FO ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT. The current study does not provide direct evidence that the C‐ring is the long‐sought pore‐forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F1FO ATP synthase dimers and involves the C‐ring.

17β-Estradiol Enhances Signalling Mediated by VEGF-A-Delta-Like Ligand 4-Notch1 Axis in Human Endothelial Cells

Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17β-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.

Electromagnetic fields counteract IL-1β activity during chondrogenesis of bovine mesenchymal stem cells

Osteoarthritis (OA) is a common joint disease associated with articular cartilage degeneration. To improve the therapeutic options of OA, tissue engineering based on the use of mesenchymal stem cells (MSCs) has emerged. However, the presence of inflammatory cytokines, such as interleukin‐1β (IL‐1β), during chondrogenesis reduces the efficacy of cartilage engineering repair procedures by preventing chondrogenic differentiation. Previous studies have shown that electromagnetic fields (EMFs) stimulate anabolic processes in OA cartilage and limit IL‐1β catabolic effects. We investigated the role of EMFs during chondrogenic differentiation of MSCs, isolated from bovine synovial fluid, in the absence and presence of IL‐1β. Pellets of MSCs were differentiated for 3 and 5 weeks with transforming growth factor‐β3 (TGFβ3), in the absence and presence of IL‐1β and exposed or unexposed to EMFs. Biochemical, quantitative real‐time RT–PCR and histological results showed that EMFs alone or in the presence of TGFβ3 play a limited role in promoting chondrogenic differentiation. Notably, in the presence of IL‐1β and TGFβ3 a recovery of proteoglycan (PG) synthesis, PG content and aggrecan and type II collagen mRNA expression in the EMF‐exposed compared to unexposed pellets was observed. Also, histological and immunohistochemical results showed an increase in staining for alcian blue, type II collagen and aggrecan in EMF‐exposed pellets. In conclusion, this study shows a significant role of EMFs in counteracting the IL‐1β‐induced inhibition of chondrogenesis, suggesting EMFs as a therapeutic strategy for improving the clinical outcome of cartilage engineering repair procedures, based on the use of MSCs. Copyright

The Role of Notch in the Cardiovascular System: Potential Adverse Effects of Investigational Notch Inhibitors

Targeting the Notch pathway is a new promising therapeutic approach for cancer patients. Inhibition of Notch is effective in the oncology setting because it causes a reduction of highly proliferative tumor cells and it inhibits survival of cancer stem cells, which are considered responsible for tumor recurrence and metastasis. Additionally, since Delta-like ligand 4 (Dll4)-activated Notch signaling is a major modulator of angiogenesis, anti-Dll4 agents are being investigated to reduce vascularization of the tumor. Notch plays a major role in the heart during the development and, after birth, in response to cardiac damage. Therefore, agents used to inhibit Notch in the tumors (gamma secretase inhibitors and anti-Dll4 agents) could potentially affect myocardial repair. The past experience with trastuzumab and other tyrosine kinase inhibitors used for cancer therapy demonstrates that the possible cardiotoxicity of agents targeting shared pathways between cancer and heart and the vasculature should be considered. To date, Notch inhibition in cancer patients has resulted only in mild gastrointestinal toxicity. Little is known about the potential long-term cardiotoxicity associated to Notch inhibition in cancer patients. In this review, we will focus on mechanisms through which inhibition of Notch signaling could lead to cardiomyocytes and endothelial dysfunctions. These adverse effects could contrast with the benefits of therapeutic responses in cancer cells during times of increased cardiac stress and/or in the presence of cardiovascular risk factor.

Biological effects of ticagrelor over clopidogrel in patients with stable coronary artery disease and chronic obstructive pulmonary disease

Summary Patients with SCAD and concomitant COPD are at high risk of cardiovascular adverse events, due to chronic inflammation, responsible of endothelial dysfunction, oxidative stress and heightened platelet reactivity (PR). The objective of this randomised clinical trial was to test if ticagrelor is superior to clopidogrel in improving endothelial function in patients with stable coronary artery disease (SCAD) and concomitant chronic obstructive pulmonary disease (COPD). Forty-six patients with SCAD and COPD undergoing percutaneous coronary intervention (PCI) were randomly assigned to receive clopidogrel (n=23) or ticagrelor (n=23) on top of standard therapy with aspirin. The following parameters were assessed at baseline and after 1 month: i) rate of apoptosis and ii) nitric oxide (NO) levels in human umbilical vein endothelial cells (HUVECs), iii) levels of reactive oxygen species (ROS) in peripheral blood mononuclear cell, iv) 29 cytokines/chemokines, v) on-treatment PR. The primary endpoint of the study was the 1-month rate of HUVECs apoptosis. The rate of apoptosis after 1 month was significantly lower in patients treated with ticagrelor (7.4 ± 1.3% vs 9.3 ± 1.5%, p<0.001), satisfying the pre-specified primary endpoint. In the ticagrelor arm, levels of NO were higher (10.1 ± 2.2 AU vs 8.5 ± 2.6 AU, p=0.03) while those of ROS (4 ± 1.8 AU vs 5.7 ± 2.8 AU, p=0.02) and P2Y 12 reactivity units (52 ± 70 PRU vs 155 ± 62 PRU, p<0.001) were lower. There were no differences in cytokines/chemokines levels and aspirin reactivity units between groups. In patients with SCAD and COPD undergoing PCI, ticagrelor, as compared to clopidogrel is superior in improving surrogate markers of endothelial function and on-treatment PR (ClinicalTrials.gov, NCT02519608). Supplementary Material to this article is available online at www.thrombosis-online.com.

Fo ATP synthase C subunit serum levels in patients with ST-segment Elevation Myocardial Infarction: Preliminary findings

BACKGROUND Recent studies in cell cultures hypothesized that the long-sought molecular pore of the mitochondrial permeability transition pore could be the Fo ATP synthase C subunit (Csub). We assessed Csub in patients with ST-segment elevation myocardial infarction (STEMI) and if it is associated with surrogate endpoints of myocardial reperfusion. METHODS We enrolled 158 first-time acute anterior STEMI treated with successful percutaneous coronary intervention (PCI). Csub was measured, after the procedure, in serum by ELISA. Csub values were related to thrombolysis in myocardial infarction (TIMI) myocardial perfusion grade (TMPG), TIMI frame count (TFC), ST-segment resolution and cardiac marker release. Echocardiography and clinical outcome were recorded at 6months. RESULTS Csub was detectable in serum and it was not normally distributed (6.3% [4-9.3%]). Csub values were higher in patients with poor values of TMPG and TFC (p=0.002 and p=0.001, respectively). Csub values were higher in patients with absent or partial ST-segment resolution as compared to those with complete ST-segment resolution (p<0.0001 and p=0.003, respectively). After adjustment for potential confounding factors, Csub emerged as an independent determinant of absent ST-segment resolution (HR 1.8, 95% CI 1.5-2.3, p=0.007), TMPG 0-1 (HR 1.7, 95% CI 1.3-2.5, p=0.01) and TFC above the median value (HR 1.5, 95% CI 1.3-2.1, p=0.03). Left ventricle ejection fraction, wall motion score index and cumulative incidence of death and heart failure were worse in patients with elevated Csub. CONCLUSIONS Our study is the first evidence that Csub is detectable in STEMI patients and that it is significantly related to several surrogate markers of myocardial reperfusion.

The role of Notch pathway in cardiovascular diseases

The recent increase in human lifespan, coupled with unhealthy diets and lifestyles have led to an unprecedented increase in cardiovascular diseases. Even in the presence of a wide range of therapeutic options with variable efficacy, mortality due to heart failure is still high and there is a need to identify new therapeutic targets. Genetic and in vitro studies have implicated the Notch signalling in the development and maintenance of the cardiovascular system through a direct effect on biological functions of vascular cells (endothelial and vascular smooth muscle cells) and cardiomyocytes. Notch signalling is also involved in the modulation of inflammation, which plays a major role in causing and exacerbating cardiovascular diseases. The Notch pathway could represent a new therapeutic target for the treatment of cardiovascular diseases.

Estrogen receptor β-dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis

Unlike age-matched men, premenopausal women benefit from cardiovascular protection. Estrogens protect against apoptosis of endothelial cells (ECs), one of the hallmarks of endothelial dysfunction leading to cardiovascular disorders, but the underlying molecular mechanisms remain poorly understood. The inflammatory cytokine TNFα causes EC apoptosis while dysregulating the Notch pathway, a major contributor to EC survival. We have previously reported that 17β-estradiol (E2) treatment activates Notch signaling in ECs. Here, we sought to assess whether in TNFα-induced inflammation Notch is involved in E2-mediated protection of the endothelium. We treated human umbilical vein endothelial cells (HUVECs) with E2, TNFα, or both and found that E2 counteracts TNFα-induced apoptosis. When Notch1 was inhibited, this E2-mediated protection was not observed, whereas ectopic overexpression of Notch1 diminished TNFα-induced apoptosis. Moreover, TNFα reduced the levels of active Notch1 protein, which were partially restored by E2 treatment. Moreover, siRNA-mediated knockdown of estrogen receptor β (ERβ), but not ERα, abolished the effect of E2 on apoptosis. Additionally, the E2-mediated regulation of the levels of active Notch1 was abrogated after silencing ERβ. In summary, our results indicate that E2 requires active Notch1 through a mechanism involving ERβ to protect the endothelium in TNFα-induced inflammation. These findings could be relevant for assessing the efficacy and applicability of menopausal hormone treatment, because they may indicate that in women with impaired Notch signaling, hormone therapy might not effectively protect the endothelium.

Mechanistic Role of mPTP in Ischemia-Reperfusion Injury.

Acute myocardial infarction (MI) is a major cause of death and disability worldwide. The treatment of choice for reducing ischemic injury and limiting infarct size (IS) in patients with ST-segment elevation MI (STEMI) is timely and effective myocardial reperfusion via primary percutaneous coronary intervention (PCI). However, myocardial reperfusion itself may induce further cardiomyocyte death, a phenomenon known as reperfusion injury (RI). The opening of a large pore in the mitochondrial membrane, namely, the mitochondrial permeability transition pore (mPTP), is widely recognized as the final step of RI and is responsible for mitochondrial and cardiomyocyte death. Although myocardial reperfusion interventions continue to improve, there remain no effective therapies for preventing RI due to incomplete knowledge regarding RI components and mechanisms and to premature translations of findings from animals to humans. In the last year, increasing amounts of data describing mPTP components, structure, regulation and function have surfaced. These data may be crucial for gaining a better understanding of RI genesis and for planning future trials evaluating new cardioprotective strategies. In this chapter, we review the role of the mPTP in RI pathophysiology and report on recent studies investigating its structure and components. Finally, we provide a brief overview of principal cardioprotective strategies and their pitfalls.

Alteration of Notch signaling and functionality of adipose tissue derived mesenchymal stem cells in heart failure

AIM Circulating mesenchymal cells increase in heart failure (HF) patients and could be used therapeutically. Our aim was to investigate whether HF affects adipose tissue derived mesenchymal cell (adMSC) isolation, functional characteristics and Notch pathway. METHODS AND RESULTS We compared 25 patients with different degrees of HF (11 NYHA classes I and II and 14 NYHA III and IV) with 10 age and gender matched controls. 100% adMSC cultures were obtained from controls, while only 72.7% and 35.7% from patients with mild or severe HF (p<0.0001). adMSC from HF patients showed higher markers of senescence (p16 positive cells: 14±2.3% in controls and 35.6±5.6% (p<0.05) and 69±14.7% (p<0.01) in mild or severe HF; γ-H2AX positive cells: 3.7±1.2%, 19.4±4.1% (p<0.05) and 23.7±3.4% (p<0.05) respectively), lower proliferation index (Ki67 positive cells: 21.5±4.9%, 13.2±2.8% and 13.7±3.2%, respectively), reduced pluripotency-associated genes (Oct4 positive cells: 86.7±4.9%, 55±12% (p<0.05) and 43.3±8.7% (p<0.05), respectively; NANOG positive cells: 89.8±3.7%, 39.6±14.4% (p<0.01) and 47±8.1%, respectively), and decreased differentiation markers (α-sarcomeric actin positive cells: 79.8±4.6%, 49±18.1% and 47±12.1% (p<0.05) and CD31-positive endothelial cells: 24.5±2.9%, 0.5±0.5% (p<0.001) and 2.3±2.3% (p<0.001), respectively). AdMSC from HF patients also showed reduced Notch transcriptional activity (lowered expression of Hey 1 and Hey 2 mRNAs). Stimulation with TNF-α of adMSC isolated from controls affected the transcription of several components of the Notch pathway (reduction of Notch 4 and Hes 1 mRNAs and increase of Notch 2 and Hey 1 mRNAs). CONCLUSIONS In HF yield and functionality of adMSC are impaired and their Notch signaling is downregulated.