Mitral regurgitation: when to intervene? : A historical perspective.

Although mitral regurgitation (MR) is the most common valvular heart disease, it should be regarded as a complex multifactorial disease that involves multiple entities. Optimal medical therapy alone does not hinder the progression of the disease, and in the 1980s it was already recognised that corrective surgery is indicated if MR is severe and patients are symptomatic (except for those with the most severe left ventricle dysfunction). Later on, asymptomatic patients with deterioration of the left ventricular ejection fraction were also operated on to avoid irreversible left ventricular dysfunction, heart failure and eventually death. However, a major drawback remains the fact that a significant group of patients is considered to have a high perioperative risk due to their advanced age or severe comorbidities. Since less invasive, percutaneous interventions have been developed and recently thoroughly investigated in the MITRA-FR and the COAPT studies, the type of intervention and also the timing have become more crucial. In this critical review of the literature, we describe what we should have learned from the past and which (haemodynamic) parameters can best predict the outcome in patients with MR.

Structure-based design of small-molecule inhibitors of EBNA1 DNA binding blocks Epstein-Barr virus latent infection and tumor growth.

Epstein-Barr virus (EBV) is a DNA tumor virus responsible for 1 to 2% of human cancers including subtypes of Burkitt's lymphoma, Hodgkin's lymphoma, gastric carcinoma, and nasopharyngeal carcinoma (NPC). Persistent latent infection drives EBV-associated tumorigenesis. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated tumors and is therefore an attractive target for therapeutic intervention. It is a multifunctional DNA binding protein critical for viral replication, genome maintenance, viral gene expression, and host cell survival. Using a fragment-based approach and x-ray crystallography, we identify a 2,3-disubstituted benzoic acid series that selectively inhibits the DNA binding activity of EBNA1. We characterize these inhibitors biochemically and in cell-based assays, including chromatin immunoprecipitation and DNA replication assays. In addition, we demonstrate the potency of EBNA1 inhibitors to suppress tumor growth in several EBV-dependent xenograft models, including patient-derived xenografts for NPC. These inhibitors selectively block EBV gene transcription and alter the cellular transforming growth factor-ß (TGF-ß) signaling pathway in NPC tumor xenografts. These EBNA1-specific inhibitors show favorable pharmacological properties and have the potential to be further developed for the treatment of EBV-associated malignancies.

Single agent and synergistic combinatorial efficacy of first-in-class small molecule imipridone ONC201 in hematological malignancies.

ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1-8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkin's lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitt's lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkin's lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.

96 perfusable blood vessels to study vascular permeability in vitro.

Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20 kDa dextran but not to 150 kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNFα and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature.

Cancer stem cell-related gene expression as a potential biomarker of response for first-in-class imipridone ONC201 in solid tumors.

Cancer stem cells (CSCs) correlate with recurrence, metastasis and poor survival in clinical studies. Encouraging results from clinical trials of CSC inhibitors have further validated CSCs as therapeutic targets. ONC201 is a first-in-class small molecule imipridone in Phase I/II clinical trials for advanced cancer. We have previously shown that ONC201 targets self-renewing, chemotherapy-resistant colorectal CSCs via Akt/ERK inhibition and DR5/TRAIL induction. In this study, we demonstrate that the anti-CSC effects of ONC201 involve early changes in stem cell-related gene expression prior to tumor cell death induction. A targeted network analysis of gene expression profiles in colorectal cancer cells revealed that ONC201 downregulates stem cell pathways such as Wnt signaling and modulates genes (ID1, ID2, ID3 and ALDH7A1) known to regulate self-renewal in colorectal, prostate cancer and glioblastoma. ONC201-mediated changes in CSC-related gene expression were validated at the RNA and protein level for each tumor type. Accordingly, we observed inhibition of self-renewal and CSC markers in prostate cancer cell lines and patient-derived glioblastoma cells upon ONC201 treatment. Interestingly, ONC201-mediated CSC depletion does not occur in colorectal cancer cells with acquired resistance to ONC201. Finally, we observed that basal expression of CSC-related genes (ID1, CD44, HES7 and TCF3) significantly correlate with ONC201 efficacy in >1000 cancer cell lines and combining the expression of multiple genes leads to a stronger overall prediction. These proof-of-concept studies provide a rationale for testing CSC expression at the RNA and protein level as a predictive and pharmacodynamic biomarker of ONC201 response in ongoing clinical studies.

ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases.

ONC201 (also called TIC10) is a small molecule that inactivates the cell proliferation- and cell survival-promoting kinases Akt and ERK and induces cell death through the proapoptotic protein TRAIL. ONC201 is currently in early-phase clinical testing for various malignancies. We found through gene expression and protein analyses that ONC201 triggered an increase in TRAIL abundance and cell death through an integrated stress response (ISR) involving the transcription factor ATF4, the transactivator CHOP, and the TRAIL receptor DR5. ATF4 was not activated in ONC201-resistant cancer cells, and in ONC201-sensitive cells, knockdown of ATF4 or CHOP partially abrogated ONC201-induced cytotoxicity and diminished the ONC201-stimulated increase in DR5 abundance. The activation of ATF4 in response to ONC201 required the kinases HRI and PKR, which phosphorylate and activate the translation initiation factor eIF2α. ONC201 rapidly triggered cell cycle arrest, which was associated with decreased abundance of cyclin D1, decreased activity of the kinase complex mTORC1, and dephosphorylation of the retinoblastoma (Rb) protein. The abundance of X-linked inhibitor of apoptosis protein (XIAP) negatively correlated with the extent of apoptosis in response to ONC201. These effects of ONC201 were independent of whether cancer cells had normal or mutant p53. Thus, ONC201 induces cell death through the coordinated induction of TRAIL by an ISR pathway.

Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53.

The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or antiangiogenesis processes. Over 50% of human cancers harbor cancer-causing mutant p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 to restore a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We demonstrate that small-molecule NSC59984 not only restores wild-type p53 signaling, but also depletes mutant p53 GOF. NSC59984 induces mutant p53 protein degradation via MDM2 and the ubiquitin-proteasome pathway. NSC59984 restores wild-type p53 signaling via p73 activation, specifically in mutant p53-expressing colorectal cancer cells. At therapeutic doses, NSC59984 induces p73-dependent cell death in cancer cells with minimal genotoxicity and without evident toxicity toward normal cells. NSC59984 synergizes with CPT11 to induce cell death in mutant p53-expressing colorectal cancer cells and inhibits mutant p53-associated colon tumor xenograft growth in a p73-dependent manner in vivo. We hypothesize that specific targeting of mutant p53 may be essential for anticancer strategies that involve the stimulation of p73 in order to efficiently restore tumor suppression. Taken together, our data identify NSC59984 as a promising lead compound for anticancer therapy that acts by targeting GOF-mutant p53 and stimulates p73 to restore the p53 pathway signaling.

Genetic and Pharmacological Screens Converge in Identifying FLIP, BCL2, and IAP Proteins as Key Regulators of Sensitivity to the TRAIL-Inducing Anticancer Agent ONC201/TIC10.

ONC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway-inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses.

The effect of fluence rate on the acute response of vessel diameter and red blood cell velocity during topical 5-aminolevulinic acid photodynamic therapy.

BACKGROUND: In a previous study it is shown that for topically applied ALA-PDT, PpIX concentration correlates with vascular changes including vasoconstriction and/or vascular leakage of small vessels and arterioles in the mouse epidermis and dermis. In this study we report on vascular responses induced by ALA-PDT for different fluence rates, including both changes in vessel diameter and dynamics in RBC velocity in arterioles, imaged using intra-vital confocal microscopy in skinfold chambers in hairless mice. Our interest is in the dynamics of vascular changes in the early stages of illumination. METHODS: We have determined the total PDT dose to be relatively low, 13 J cm(-2), and fluence rates of 26, 65 and 130 mW cm(-2) were investigated. Local vascular effects occurred very soon after the start of the therapeutic illumination in ALA-PDT. RESULTS: In this study, we did not find a significant difference between fluence rates. Arterioles were particularly sensitive to vasoconstriction during low dose PDT, often resulting in complete vasoconstriction. When we observed complete vasoconstriction, this coincided with changes in RBC velocity. CONCLUSION: Since the therapeutic effects of PDT are dependent on a fine balance between the need for oxygen during illumination and disruption of the vasculature, the results of the present study add to our understanding of acute vascular effects during ALA-PDT and aid our efforts to optimize PDT using porphyrin pre-cursors.

Targeting tumor suppressor p53 for cancer therapy: strategies, challenges and opportunities.

p53 is one of the most important tumor suppressor genes that is frequently mutated in human cancers. Generally, p53 functions as a transcription factor that is stabilized and activated by various genotoxic and cellular stress signals, such as DNA damage, hypoxia, oncogene activation and nutrient deprivation, consequently leading to cell cycle arrest, apoptosis, senescence and metabolic adaptation. p53 not only becomes functionally deficient in most cancers, but not infrequently mutant p53 also acquires dominant negative activity and oncogenic properties. p53 has remained an attractive target for cancer therapy. Strategies targeting p53 have been developed including gene therapy to restore p53 function, inhibition of p53-MDM2 interaction, restoration of mutant p53 to wild-type p53, targeting p53 family proteins, eliminating mutant p53, as well as p53-based vaccines. Some of these p53-targeted therapies have entered clinical trials. We discuss the therapeutic potential of p53, with particular focus on the therapeutic strategies to rescue p53 inactivation in human cancers. In addition, we discuss the challenges of p53-targeted therapy and new opportunities for the future.

Prodigiosin rescues deficient p53 signaling and antitumor effects via upregulating p73 and disrupting its interaction with mutant p53.

p53 reactivation offers a broad-based strategy for cancer therapy. In this study, we report the identification of prodigiosin that can reactivate p53 family-dependent transcriptional activity in p53-deficient human colon cancer cells. Prodigiosin and its structural analogue (compound R) induced the expression of p53 target genes accompanied by cell-cycle arrest and apoptosis in p53-deficient cancer cells. Prodigiosin restored p53 signaling in cancer cells harboring hotspot TP53 mutations, with little to no detectable cytotoxicity in normal human fibroblasts and with no genotoxicity. Prodigiosin induced the expression of p73 and disrupted its interaction with mutant p53, thereby rescuing p53 pathway deficiency and promoting antitumor effects. The disruption of mutant p53/p73 interaction was specific to prodigiosin and not related to mTOR inhibition. Our findings suggest that mutant p53 needs to be targeted in the context of p73 stimulation to allow efficient restoration of the p53 pathway. In exhibiting this capability, prodigiosin and its analogue provide lead compounds to rescue deficiencies in the p53 pathway in cancer cells by upregulating p73 and targeting mutant p53/p73 interaction there.

Antithrombotic therapy in patients undergoing TAVI: an overview of Dutch hospitals.

PURPOSE: To assess current antithrombotic treatment strategies in the Netherlands in patients undergoing transcatheter aortic valve implantation (TAVI). METHODS: For every Dutch hospital performing TAVI (n = 14) an interventional cardiologist experienced in performing TAVI was interviewed concerning heparin, aspirin, thienopyridine and oral anticoagulation treatment in patients undergoing TAVI. RESULTS: The response rate was 100 %. In every centre, a protocol for antithrombotic treatment after TAVI was available. Aspirin was prescribed in all centres, concomitant clopidogrel was prescribed 13 of the 14 centres. Duration of concomitant clopidogrel was 3 months in over two-thirds of cases. In 2 centres, duration of concomitant clopidogrel was based upon type of prosthesis: 6 months versus 3 months for supra-annular and intra-annular prostheses, respectively. CONCLUSIONS: Leaning on a small basis of evidence and recommendations, the antithrombotic policy for patients undergoing TAVI is highly variable in the Netherlands. As a standardised regimen might further reduce haemorrhagic complications, large randomised clinical trials may help to establish the most appropriate approach.

The effect of light fractionation with a 2-h dark interval on the efficacy of topical hexyl-aminolevulinate photodynamic therapy in normal mouse skin.

BACKGROUND: Light fractionation with a 2-h dark interval increases the efficacy of topical aminolevulinic acid (ALA) photodynamic therapy (PDT). Hexyl-aminolevulinate (HAL) is the hexyl ester of ALA. Both HAL and ALA lead to protoporphyrin IX (PpIX) accumulation in endothelial cells and to vascular effects, which are important for light fractionation. We investigated light fractionation for HAL-PDT in a mouse skin model and compared this with ALA. METHODS: Three illumination schemes were studied: (a) 100 J cm(-2) in a single illumination; (b) 50+50 J cm(-2) in a twofold illumination; (c) a small first light fraction until 50% of PpIX was photobleached (ca. 3 J cm(-2)), followed by 97 J cm(-2) 2h later. PpIX fluorescence was measured continuously during illumination. Efficacy was evaluated by daily visual skin damage scoring up to 7 days after PDT. RESULTS: Light fractionation showed a trend towards increased efficacy for HAL-PDT. Both the initial PpIX synthesis and the PpIX resynthesis during the dark interval were higher for ALA, but these were not correlated with efficacy. Single HAL-PDT was more effective than single ALA-PDT. Photobleaching rates of HAL and ALA were similar indicating similar biodistributions at depth. CONCLUSION: Our results provide evidence to support that light fractionation may be beneficial for HAL-PDT. We are cautious because we found only a non-significant increase in response. However, combining our results with literature data suggest that the illumination scheme may be further optimized for HAL-PDT to potentially enhance the effect of light fractionation.

Topical hexylaminolevulinate and aminolevulinic acid photodynamic therapy: complete arteriole vasoconstriction occurs frequently and depends on protoporphyrin IX concentration in vessel wall.

Vascular responses to photodynamic therapy (PDT) may influence the availability of oxygen during PDT and the extent of tumor destruction after PDT. However, for topical PDT vascular effects are largely unknown. Arteriole and venule diameters were measured before and after hexylaminolevulinate (HAL) and aminolevulinic acid (ALA) PDT and related to the protoporphyrin IX (PpIX) concentration in the vessel wall. A mouse skin fold chamber model and an intravital confocal microscope allowed direct imaging of the subcutaneous vessels underlying the treated area. In both HAL and ALA groups over 60% of arterioles constricted completely, while venules generally did not respond, except for two larger veins that constricted partially. Arteriole vasoconstriction strongly correlated with PpIX fluorescence intensity in the arteriole wall. Total PpIX fluorescence intensity was significantly higher for HAL than ALA for the whole area that was imaged but not for the arteriole walls. In conclusion, complete arteriole vasoconstriction occurs frequently in both HAL and ALA based topical PDT, especially when relatively high PpIX concentrations in arteriole walls are reached. Vasoconstriction will likely influence PDT effect and should be considered in studies on topical HAL and ALA-PDT. Also, our results may redefine the vasculature as a potential secondary target for topical PDT.

In vivo quantification of the scattering properties of tissue using multi-diameter single fiber reflectance spectroscopy.

Multi diameter single fiber reflectance (MDSFR) spectroscopy is a non-invasive optical technique based on using multiple fibers of different diameters to determine both the reduced scattering coefficient (µs') and a parameter γ that is related to the angular distribution of scattering, where γ = (1-g2)/(1-g1) and g1 and g2 the first and second moment of the phase function, respectively. Here we present the first in vivo MDSFR measurements of µs'(λ) and γ(λ) and their wavelength dependence. MDSFR is performed on nineteen mice in four tissue types including skin, liver, normal tongue and in an orthotopic oral squamous cell carcinoma. The wavelength-dependent slope of µs'(λ) (scattering power) is significantly higher for tongue and skin than for oral cancer and liver. The reduced scattering coefficient at 800 nm of oral cancer is significantly higher than of normal tongue and liver. Gamma generally increases with increasing wavelength; for tumor it increases monotonically with wavelength, while for skin, liver and tongue γ(λ) reaches a plateau or even decreases for longer wavelengths. The mean γ(λ) in the wavelength range 400-850 nm is highest for liver (1.87 ± 0.07) and lowest for skin (1.37 ± 0.14). Gamma of tumor and normal tongue falls in between these values where tumor exhibits a higher average γ(λ) (1.72 ± 0.09) than normal tongue (1.58 ± 0.07). This study shows the potential of using light scattering spectroscopy to optically characterize tissue in vivo.

Analysis of glutamine dependency in non-small cell lung cancer: GLS1 splice variant GAC is essential for cancer cell growth.

One of the hallmarks of cancer is metabolic deregulation. Many tumors display increased glucose uptake and breakdown through the process of aerobic glycolysis, also known as the Warburg effect. Less studied in cancer development and progression is the importance of the glutamine (Gln) pathway, which provides cells with a variety of essential products to sustain cell proliferation, such as ATP and macromolecules for biosynthesis. To this end Gln dependency was assessed in a panel of non-small cell lung cancer lines (NSCLC). Gln was found to be essential for the growth of cells with high rates of glutaminolysis, and after exploring multiple genes in the Gln pathway, GLS1 was found to be the key enzyme associated with this dependence. This dependence was confirmed by observing the rescue of decreased growth by exogenous addition of downstream metabolites of glutaminolysis. Expression of the GLS1 splice variant KGA was found to be decreased in tumors compared with normal lung tissue. Transient knock down of GLS1 splice variants indicated that loss of GAC had the most detrimental effect on cancer cell growth. In conclusion, NSCLC cell lines depend on Gln for glutaminolysis to a varying degree, in which the GLS1 splice variant GAC plays an essential role and is a potential target for cancer metabolism-directed therapy.

Planning and monitoring of patients for electrical cardioversion for atrial fibrillation.

OBJECTIVES: This study evaluated the waiting list for elective electrical cardioversion (ECV) for persistent atrial fibrillation (AF), focusing on when and why procedures were postponed. We compared the effects of management of the waiting list conducted by physicians versus management by nurse practitioners (NPs) and we evaluated the safety of our anticoagulating policy by means of bleeding or thromboembolic complications during and after ECV. BACKGROUND: Not all patients selected for ECV receive their treatment at the first planned instance due to a variety of reasons. These reasons are still undocumented. METHODS: We evaluated 250 consecutive patients with persistent AF admitted to our clinic for elective ECV. RESULTS: Within 5 to 6 weeks, 186 of 242 patients (77%) received ECV. The main reason for postponing an ECV was an inadequate international normalised ratio (INR); other reasons included spontaneous sinus rhythm and switch to rate control. A total of 23 of the 147 patients (16%) managed by the research physician were postponed due to an inadequate INR at admission versus 4 out of 98 patients (4%) managed by NPs (p = 0.005) CONCLUSION: An inadequate INR is the main reason for postponing an ECV. Management of ECV by NPs is safe and leads to less postponing on admission.

Novel common integration sites targeted by mouse mammary tumor virus insertion in mammary tumors have oncogenic activity.

Non-acute transforming retroviruses like mouse mammary tumor virus (MMTV) cause cancer, at least in part, through integration near cellular genes involved in growth control, thereby de-regulating their expression. It is well-established that MMTV commonly integrates near and activates expression of members of the Wnt and Fgf pathways in mammary tumors. However, there are a significant number of tumors for which the proviral integration sites have not been identified. Here, we used high through-put screening to identify common integration sites (CISs) in MMTV-induced tumors from C3H/HeN and BALB/c mice. As expected, members of both the Wnt and Fgf families were identified in this screen. In addition, a number of novel CISs were found, including Tcf7l2, Antxr1/Tem8, and Arhgap18. We show here that expression of these three putative oncogenes in normal murine mammary gland cells altered their growth kinetics and caused their morphological transformation when grown in three dimensional cultures. Additionally, expression of Tcf7l2 and Antxr1/Tem8 sensitized cells to exogenous WNT ligand. As Tcf7l2, Antxr1/Tem8, and Arhgap18 have been associated with human breast and other cancers, these data demonstrate that MMTV-induced insertional mutation remains an important means for identifying genes involved in breast cancer.