Ageing, hypertension and aortic valve stenosis - Understanding the series circuit using cardiac magnetic resonance and applanation tonometry.

BACKGROUND: Aortic stenosis (AS) is no longer considered to be a disease of fixed left ventricular (LV) afterload, but rather, functions as a series circuit, with important contributions from both the valve and vasculature. Patients with AS are typically elderly, with hypertension and a markedly remodelled aorta. The arterial component is sizeable, and yet, quantifying this to-date has been difficult to determine. We compared measurement of aortic pressure, flow and global LV load using a cardiac magnetic resonance (CMR)/applanation tonometry (AT) technique to uncouple ventriculo-arterial (VA) interactions. METHODS: 20 healthy elderly patients and 20 with AS underwent a CMR/AT protocol. CMR provided LV volume and aortic flow simultaneously with AT pressure acquisition. Aortic pressure was derived by transformation of the AT waveform. Systemic vascular resistance (SVR) and global LV load were determined as the relationship of pressure to flow in the frequency domain. Values from both cohorts were compared. RESULTS: AS patients were older (p â€‹< â€‹0.01) albeit with no significant difference in brachial or central aortic pressure. SVR (14228 vs 19906 â€‹dyne â€‹s.cm-3; p â€‹= â€‹0.02) and load (740 vs 946 â€‹dyne â€‹s.cm-3; p â€‹= â€‹0.02) were higher in patients with AS, whilst aortic peak flow velocity was lower (38 vs 58 â€‹cm/s; p â€‹< â€‹0.01). CONCLUSIONS: Quantification of aortic pressure, flow velocity and global LV load using a simultaneous CMR/AT technique is able to demonstrate the progressive effects of hypertension and aortic stiffening with advanced age and valvular stenosis. This technique may help to better identify future patients at risk of VA coupling mismatch after correction of AS.

Enhancing venetoclax activity in acute myeloid leukemia by co-targeting MCL1.

Targeted therapies are frequently combined with standard cytotoxic drugs to enhance clinical response. Targeting the B-cell lymphoma 2 (BCL-2) family of proteins is an attractive option to combat chemoresistance in leukemia. Preclinical and clinical studies indicate modest single-agent activity with selective BCL-2 inhibitors (for example, venetoclax). We show that venetoclax synergizes with cytarabine and idarubicin to increase antileukemic efficacy in a TP53-dependent manner. Although TP53 deficiency impaired sensitivity to combined venetoclax and chemotherapy, higher-dose idarubicin was able to suppress MCL1 and induce cell death independently of TP53. Consistent with an MCL1-specific effect, cell death from high-dose idarubicin was dependent on pro-apoptotic Bak. Combining higher-dose idarubicin with venetoclax was able to partially overcome resistance in Bak-deficient cells. Using inducible vectors and venetoclax to differentially target anti-apoptotic BCL-2 family members, BCL-2 and MCL1 emerged as critical and complementary proteins regulating cell survival in acute myeloid leukemia. Dual targeting of BCL-2 and MCL1, but not either alone, prolonged survival of leukemia-bearing mice. In conclusion, our findings support the further investigation of venetoclax in combination with standard chemotherapy, including intensified doses of idarubicin. Venetoclax should also be investigated in combination with direct inhibitors of MCL1 as a chemotherapy-free approach in the future.

Primary central nervous system posttransplantation lymphoproliferative disorder after heart and lung transplantation.

Primary central nervous system posttransplantation lymphoproliferative disorder (PCNS-PTLD) is uncommon, especially after heart or lung transplantation. Database analysis from a single heart and lung transplantation centre and a literature review pertaining to PCNS-PTLD was performed. In this study, the prevalence of PCNS-PTLD was 0.18% after heart and/or lung transplants. Of 1674 transplants, three cases of PCNS-PTLD developed 14 months, 9 years and 17 years posttransplant, and all were Epstein-Barr virus driven malignancies. Literature review of the topic revealed predominantly retrospective studies, with most reported cases after renal transplantation. The overall survival is poor, and it may be improved by early diagnosis and treatment. There are no published guidelines on the management of PCNS-PTLD; immune-chemotherapy in conjunction with reduction of immune suppression is preferred based on available evidence.

Potential role of coenzyme Q10 in facilitating recovery from statin-induced rhabdomyolysis.

Rhabdomyolysis is a rare, but serious complication of statin therapy, and represents the most severe end of the spectrum of statin-induced myotoxicity. We report a case where coenzyme Q10 facilitated recovery from statin-induced rhabdomyolysis and acute renal failure, which had initially persisted despite statin cessation and haemodialysis. This observation is biologically plausible due to the recognised importance of coenzyme Q10 in mitochondrial bioenergetics within myocytes, and the fact that statins inhibit farnesyl pyrophosphate production, a biochemical step crucial for coenzyme Q10 synthesis. Coenzyme Q10 is generally well tolerated, and may potentially benefit patients with statin-induced rhabdomyolysis.

Normothermic ex vivo perfusion provides superior organ preservation and enables viability assessment of hearts from DCD donors.

The shortage of donors in cardiac transplantation may be alleviated by the use of allografts from donation after circulatory death (DCD) donors. We have previously shown that hearts exposed to 30 min warm ischemic time and then flushed with Celsior supplemented with agents that activate ischemic postconditioning pathways, show complete recovery on a blood-perfused ex vivo working heart apparatus. In this study, these findings were assessed in a porcine orthotopic heart transplant model. DCD hearts were preserved with either normothermic ex vivo perfusion (NEVP) using a clinically approved device, or with standard cold storage (CS) for 4 h. Orthotopic transplantation into recipient animals was subsequently undertaken. Five of six hearts preserved with NEVP demonstrated favorable lactate profiles during NEVP and all five could be weaned off cardiopulmonary bypass posttransplant, compared with 0 of 3 hearts preserved with CS (p < 0.05, Fisher's exact test). In conclusion, DCD hearts flushed with supplemented Celsior solution and preserved with NEVP display viability before and after transplantation. Viability studies of human DCD hearts using NEVP are warranted.

Predictors of paravalvular aortic regurgitation following self-expanding Medtronic CoreValve implantation: the role of annulus size, degree of calcification, and balloon size during pre-implantation valvuloplasty and implant depth.

OBJECTIVES: We sought to investigate the role of balloon size during pre-implantation valvuloplasty in predicting AR and optimal Medtronic CoreValve (MCS) implantation depth. BACKGROUND: Paravalvular aortic regurgitation (AR) is common following MCS implantation. A number of anatomical and procedural variables have been proposed as determinants of AR including degree of valve calcification, valve undersizing and implantation depth. METHODS: We conducted a multicenter retrospective analysis of 282 patients who had undergone MCS implantation with prior cardiac CT annular sizing between 2007 and 2011. Native valve minimum (Dmin), maximum (Dmax) and arithmetic mean (Dmean) annulus diameters as well as agatston calcium score were recorded. Nominal and achieved balloon size was also recorded. AR was assessed using contrast angiography at the end of each procedure. Implant depth was measured as the mean distance from the nadir of the non- and left coronary sinuses to the distal valve frame angiographically. RESULTS: 29 mm and 26 mm MCS were implanted in 60% and 39% of patients respectively. The majority of patients (N=165) developed AR <2 following MCS implantation. AR ≥3 was observed in 16% of the study population. High agatston calcium score and Dmean were found to be independent predictors of AR ≥3 in multivariate analysis (P<0.0001). Nominal balloon diameter and the number of balloon inflations did not influence AR. However a small achieved balloon diameter-to-Dmean ratio (≤0.85) showed modest correlation with AR ≥3 (P=0.04). This observation was made irrespective of the degree of valve calcification. A small MCS size-to-Dmean ratio is also associated with AR ≥3 (P=0.001). A mean implantation depth of ≥8+2mm was also associated with AR ≥3. Implantation depth of ≥12 mm was associated with small MCS diameter-to-Dmean ratio and increased 30-day mortality. CONCLUSION: CT measured aortic annulus diameter and agatston calcium score remain important predictors of significant AR. Other procedural predictors include valve undersizing and low implantation depth. A small achieved balloon diameter-to-Dmean ratio might also predict AR ≥3. Our findings confirm that a small achieved balloon size during pre-implantation valvuloplasty predicts moderate-severe AR in addition to previously documented factors.

Increasing the tolerance of DCD hearts to warm ischemia by pharmacological postconditioning.

Donation after circulatory death (DCD) offers a potential additional source of cardiac allografts. We used a porcine asphyxia model to evaluate viability of DCD hearts subjected to warm ischemic times (WIT) of 20­40 min prior to flushing with Celsior (C) solution. We then assessed potential benefits of supplementing C with erythropoietin, glyceryl trinitrate and zoniporide (Cs), a combination that we have shown previously to activate ischemic postconditioning pathways. Hearts flushed with C/Cs were assessed for functional, biochemical and metabolic recovery on an ex vivo working heart apparatus. Hearts exposed to 20-min WIT showed full recovery of functional and metabolic profiles compared with control hearts (no WIT). Hearts subjected to 30- or 40-min WIT prior to C solution showed partial and no recovery, respectively. Hearts exposed to 30-min WIT and Cs solution displayed complete recovery, while hearts exposed to 40-min WIT and Cs solution demonstrated partial recovery. We conclude that DCD hearts flushed with C solution demonstrate complete recovery up to 20-min WIT after which there is rapid loss of viability. Cs extends the limit of WIT tolerability to 30 min. DCD hearts with ≤30-min WIT may be suitable for transplantation and warrant assessment in a transplant model.

Hoxb8 regulates expression of microRNAs to control cell death and differentiation.

Hoxb8 overexpression immortalises haematopoietic progenitor cells in a growth-factor-dependant manner and co-operates with interleukin-3 (IL-3) to cause acute myeloid leukaemia. To further understand how Hoxb8 contributes to myeloid cell immortalisation, we generated IL-3-dependant myeloid cells expressing Hoxb8 under the control of an inducible promoter. Downregulation of Hoxb8, in the presence of IL-3, caused cell-cycle arrest and apoptosis in the majority of cells. Apoptosis was dependant on Bax and Bak and, in part, on Bim, which was repressed by Hoxb8. Deletion of the miR-17∼92 seed sequences in the Bim 3'UTR abolished Hoxb8-dependant regulation of Bim reporter constructs. Expression of all six miRNAs from this cluster were elevated when Hoxb8 was overexpressed. The miR-17∼92 cluster was required for repression of Bim in Hoxb8-immortalised cells and deletion of the miR-17∼92 cluster substantially inhibited Hoxb8, but not Hoxa9, mediated survival and proliferation. Hoxb8 appears to promote miR-17∼92 expression through c-Myc, a known transcriptional regulator of the miR-17∼92 cluster. We have uncovered a previously unrecognised link between Hoxb8 expression and microRNAs that provides a new insight into the oncogenic functions of Hoxb8.

Akt1 is the principal Akt isoform regulating apoptosis in limiting cytokine concentrations.

The activation of the Akt signalling in response to cytokine receptor signalling promotes protein synthesis, cellular growth and proliferation. To determine the role of Akt in interleukin-3 (IL-3) signalling, we generated IL-3-dependent myeloid cell lines from mice lacking Akt1, Akt2 or Akt3. Akt1 deletion resulted in accelerated apoptosis at low concentrations of IL-3. Expression of constitutively active Akt1 was sufficient to delay apoptosis in response to IL-3 withdrawal, but not sufficient to induce proliferation in the absence of IL-3. Akt1 prolonged survival of Bim- or Bad-deficient cells, but not cells lacking Puma, indicating that Akt1-dependent repression of apoptosis was in part dependent on Puma and independent of Bim or Bad. Our data show that a key role of Akt1 during IL-3 signalling is to repress p53-dependent apoptosis pathways, including transcriptional upregulation of Puma. Moreover, our data indicate that regulation of BH3-only proteins by Akt is dispensable for Akt-dependent cell survival.

Enhanced preservation of pig cardiac allografts by combining erythropoietin with glyceryl trinitrate and zoniporide.

Erythropoietin has a tissue-protective effect independent of its erythropoietic effect that may be enhanced by combining it with the nitric oxide donor glyceryl trinitrate (GTN) and the sodium-hydrogen exchange inhibitor zoniporide in rat hearts stored with an extracellular-based preservation solution (EBPS). We thus sought to test this combination of agents in a porcine model of orthotopic heart transplantation incorporating donor brain death and total ischaemic time of approximately 260 min. Pig hearts were stored in one of four storage solutions: unmodified EBPS (CON), EBPS supplemented with GTN and zoniporide (GZ), EBPS supplemented with erythropoietin and zoniporide (EZ), or EBPS supplemented with all three agents (EGZ). A total of 4/5 EGZ hearts were successfully weaned from cardiopulmonary bypass compared with only 2/5 GZ hearts, 0/5 CON hearts and 0/5 EG hearts (p = 0.017). Following weaning from bypass EGZ hearts demonstrated superior contractility and haemodynamics than GZ hearts. All weaned hearts displayed impaired diastolic function. Release of troponin I from EGZ hearts was lower than all other groups. In conclusion, supplementation of EBPS with erythropoietin, glyceryl trinitrate and zoniporide provided superior donor heart preservation than all other strategies tested.

Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death.

P53-upregulated modifier of apoptosis (PUMA), a pro-apoptotic member of the Bcl-2 family, is transcriptionally activated by p53 and is a key effector of p53-dependent apoptosis. We show that PUMA protein is subject to rapid post-translational regulation by phosphorylation at a conserved residue, serine 10, following serum or interleukin-3 (IL-3) stimulation. Serine 10 is not within the Bcl-2 homology (BH3) domain, and PUMA phosphorylated at serine 10 retained the ability to co-immunoprecipitate with antiapoptotic Bcl-2 family members. However, phosphorylated PUMA was targeted for proteasomal degradation indicating that it is less stable than unphosphorylated PUMA. Importantly, we identified IKK1/IKK2/Nemo as the kinase complex that interacts with and phosphorylates PUMA, thereby also demonstrating that IL-3 activates NFκB signaling. The identification and characterization of this novel survival pathway has important implications for IL-3 signaling and hematopoietic cell development.

Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim.

Bcl-2 family members regulate apoptosis in response to cytokine withdrawal and a broad range of cytotoxic stimuli. Pro-apoptotic Bcl-2 family members Bax and Bak are essential for apoptosis triggered by interleukin-3 (IL-3) withdrawal in myeloid cells. The BH3-only protein Puma is critical for initiation of IL-3 withdrawal-induced apoptosis, because IL-3-deprived Puma(-/-) cells show increased capacity to form colonies when IL-3 is restored. To investigate the mechanisms of Puma-induced apoptosis and the interactions between Puma and other Bcl-2 family members, we expressed Puma under an inducible promoter in cells lacking one or more Bcl-2 family members. Puma rapidly induced apoptosis in cells lacking the BH3-only proteins, Bid and Bim. Puma expression resulted in activation of Bax, but Puma killing was not dependent on Bax or Bak alone as Puma readily induced apoptosis in cells lacking either of these proteins, but could not kill cells deficient for both. Puma co-immunoprecipitated with the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1 but not with Bax or Bak. These data indicate that Puma functions, in the context of induced overexpression or IL-3 deprivation, primarily by binding and inactivating anti-apoptotic Bcl-2 family members.

Oxazaborolidine derivatives inducing autoinducer-2 signal transduction in Vibrio harveyi.

The bioluminescence of the marine bacterium Vibrio harveyi is controlled by quorum sensing. This effect is mediated by production, accumulation, and auto-detection of the species-specific autoinducer 1 (AI-1), autoinducer 2 (AI-2), and the V. cholerae autoinducer 1 (CAI-1). The V. harveyi AI-2 was recently identified as furanosyl borate diester. We synthesized several oxazaborolidine derivatives that chemically resemble the structure of AI-2. Five oxazaborolidine derivatives (BNO-1 to BNO-5) were tested, however only BNO-1 (3,4-dimethyl-2,5-diphenyl-1,3,2-oxazaborolidine), and BNO-5 (2-butyl-3,4-dimethyl-5-phenyl-1,3,2-oxazaborolidine) strongly induced V. harveyi bioluminescence in V. harveyi mutant (BB170) lacking sensor 1. A dose-dependent relationship between those oxazaborolidine derivatives and bioluminescence induction was observed with this V. harveyi strain (BB170). BNO-1 and BNO-5 did not affect V. harveyi BB886 lacking sensor 2. Using a mutant strain which produces neither AI-1 nor AI-2 (V. harveyi MM77) we showed that the presence of spent medium containing AI-2 is essential for BNO-1 and BNO-5 activity. This effect was similar when introducing the spent medium and the BNOs together or at a 3-h interval. A comparable induction of bioluminescence was observed when using synthetic DPD (pre-AI-2) in the presence of BNO-1 or BNO-5. The mode of action of BNO-1 and BNO-5 on bioluminescence of V. harveyi is of a co-agonist category. BNO-1 and BNO-5 enhanced AI-2 signal transduction only in the presence of AI-2 and only via sensor 2 cascade. BNO-1 and BNO-5 are the first oxazaborolidines reported to affect AI-2 activity. Those derivatives represent a new class of borates which may become prototypes of novel agonists of quorum sensing mediated by AI-2 in V. harveyi.

The Caenorhabditis elegans CED-9 protein does not directly inhibit the caspase CED-3, in vitro nor in yeast.

A genetically defined pathway orchestrates the removal of 131 of the 1090 somatic cells generated during the development of the hermaphrodite nematode Caenorhabditis elegans. Regulation of apoptosis is highly evolutionarily conserved and the nematode cell death pathway is a valuable model for studying mammalian apoptotic pathways, the dysregulation of which can contribute to numerous diseases. The nematode caspase CED-3 is ultimately responsible for the destruction of worm cells in response to apoptotic signals, but it must first be activated by CED-4. CED-9 inhibits programmed cell death and considerable data have demonstrated that CED-9 can directly bind and inhibit CED-4. However, it has been suggested that CED-9 may also directly inhibit CED-3. In this study, we used a yeast-based system and biochemical approaches to explore this second potential mechanism of action. While we confirmed the ability of CED-9 to inhibit CED-4, our data argue that CED-9 can not directly inhibit CED-3.

The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis.

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the P1 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide-substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

Simultaneous analysis of surface marker expression and cell cycle progression in human peripheral blood mononuclear cells.

One method for examining cell cycle kinetics by flow cytometry uses continuous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA fluorochrome Hoechst 33258. After counterstaining with a secondary DNA fluorochrome (e.g., ethidium bromide), the analyst can distinguish cells in different phases of the cell cycle over a number of mitotic cycles with flow cytometry. In this report, we describe a modification of the flow cytometric BrdU-Hoechst assay that allows combined analysis of cell proliferation and immunophenotyping at the single cell level. To demonstrate an application of this method, human peripheral blood mononuclear cells were stimulated with tetanus toxoid or interleukin-2 for up to 6 days in the presence of BrdU, harvested, and immunostained for the cell surface markers CD3, CD4, CD8, CD14, CD19, and the cytokine receptor, CCR5. We used four-color flow cytometry analyses to simultaneously measure cell proliferation and surface marker expression, for the purpose of immunophenotyping and identifying specific cell subsets responding to antigen stimulation. Our successful application of this method suggests that it may be used to study immune responses at the molecular and cellular level and to identify mechanisms of immune system modulation.