Foam fractionation studies of recombinant human apolipoprotein A-I.

Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.

High platelet adrenergic activity and concomitant activation of the pituitary/medullar axis as alarming laboratory parameters in ACS survivors-the STRESS-AMI study.

Introduction: Kinetics of stress-related biological parameters were determined in acute coronary syndrome (ACS) patients undergoing complex cardiovascular rehabilitation. Methods: We determined platelet functionality in the absence/presence of a selective alpha-2 adrenergic receptor inhibitor, atipemazole parallel with salivary cortisol levels at enrolment, and at 3- and 12-months follow-up in 75 ACS patients with percutaneous coronary intervention. Results: Pharmacological/non-pharmacological secondary prevention methods have been efficiently applied. Baseline aggregometry indicated platelet hyperactivity, decreasing gradually and being significantly reduced late, at 12 months (p < 0.05). Cortisol levels followed similar kinetics (p < 0.05). Baseline epinephrine-induced aggregations (EIA) significantly correlated with most of the other platelet agonists, even at subsequent time-points. Patients with upper-quartile EIA at enrolment (EIA-UQ) had significantly higher ADP- and collagen-induced aggregations at enrolment, at 3- and 12-months follow-up as well, indicating that high adrenergic response in the acute phase is accompanied by general platelet hyperactivity and predicts sustained platelet activation. In the EIA-UQ group higher cardiac biomarker release, elevated C-reactive protein and cortisol levels, and lower baseline left ventricular ejection fraction were detected.Atipemazole significantly reduced platelet aggregation induced by several platelet agonists, being most potent and comparable to full in vitro P2Y12 inhibition on collagen-induced aggregations (p < 0.05), indicating that catecholamines might serve as promt/long-term modulators of platelet function. Discussion: Despite effective CCR programme and dual antiplatelet therapy, prolonged activation of sympathetic neuroendocrine system and general platelet hyperactivity can be detected up to one year in ACS patients with high adrenergic platelet activity. Moreover, initial high adrenergic activity is accompanied by clinical parameters associated to increased cardiovascular risk, therefore early identification of these patients might support complex optimal long-term therapy.

The effect of kidney function on guideline-directed medical therapy implementation and prognosis in heart failure with reduced ejection fraction.

BACKGROUND: Kidney dysfunction (KD) is a main limiting factor of applying guideline-directed medical therapy (GDMT) and reaching the recommended target doses (TD) in heart failure (HF) with reduced ejection fraction (HFrEF). HYPOTHESIS: We aimed to assess the success of optimization, long-term applicability, and adherence of neurohormonal antagonist triple therapy (TT:RASi [ACEi/ARB/ARNI] + ßB + MRA) according to the KD after a HF hospitalization and to investigate its impact on prognosis. METHODS: The data of 247 real-world, consecutive patients were analyzed who were hospitalized in 2019-2021 for HFrEF and then were followed-up for 1 year. The application and the ratio of reached TD of TT at hospital discharge and at 1 year were assessed comparing KD categories (eGFR: ≥90, 60-89, 45-59, 30-44, <30 mL/min/1.73 m2 ). Moreover, 1-year all-cause mortality and rehospitalization rates in KD subgroups were investigated. RESULTS: Majority of the patients received TT at hospital discharge (77%) and at 1 year (73%). More severe KD led to a lower application ratio (p < .05) of TT (92%, 88%, 80%, 73%, 31%) at discharge and at 1 year (81%, 76%, 76%, 68%, 40%). Patients with more severe KD were less likely (p < .05) to receive TD of MRA (81%, 68%, 78%, 61%, 52%) at discharge and a RASi (53%, 49%, 45%, 21%, 27%) at 1 year. One-year all-cause mortality (14%, 15%, 16%, 33%, 48%, p < .001), the ratio of all-cause rehospitalizations (30%, 35%, 40%, 43%, 52%, p = .028), and rehospitalizations for HF (8%, 13%, 18%, 20%, 38%, p = .001) were significantly higher in more severe KD categories. CONCLUSIONS: KD unfavorably affects the application of TT in HFrEF, however poorer mortality and rehospitalization rates among them highlight the role of the conscious implementation and up-titration of GDMT.

The Impact of Specialised Heart Failure Outpatient Care on the Long-Term Application of Guideline-Directed Medical Therapy and on Prognosis in Heart Failure with Reduced Ejection Fraction.

(1) Background: Besides the use of guideline-directed medical therapy (GDMT), multidisciplinary heart failure (HF) outpatient care (HFOC) is of strategic importance in HFrEF. (2) Methods: Data from 257 hospitalised HFrEF patients between 2019 and 2021 were retrospectively analysed. Application and target doses of GDMT were compared between HFOC and non-HFOC patients at discharge and at 1 year. 1-year all-cause mortality (ACM) and rehospitalisation (ACH) rates were compared using the Cox proportional hazard model. The effect of HFOC on GDMT and on prognosis after propensity score matching (PSM) of 168 patients and the independent predictors of 1-year ACM and ACH were also evaluated. (3) Results: At 1 year, the application of RASi, MRA and triple therapy (TT: RASi + ßB + MRA) was higher (p < 0.05) in the HFOC group, as was the proportion of target doses of ARNI, ßB, MRA and TT. After PSM, the composite of 1-year ACM or ACH was more favourable with HFOC (propensity-adjusted HR = 0.625, 95% CI = 0.401-0.974, p = 0.038). Independent predictors of 1-year ACM were age, systolic blood pressure, application of TT and HFOC, while 1-year ACH was influenced by the application of TT. (4) Conclusions: HFOC may positively impact GDMT use and prognosis in HFrEF even within the first year of its initiation.

PCSK7: A novel regulator of apolipoprotein B and a potential target against non-alcoholic fatty liver disease.

BACKGROUND: Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS: We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS: Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and ß-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS: Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.

Compreendendo a tumorigênese das neoplasias astrocitárias / Understanding the astrocytic neoplasm tumorigenesis

As neoplasias astrocitárias correspondem a 60 por cento dos tumores do sistema nervoso central, sendo o estudo da biologia molecular importante passo para a compreensão da gênese e comportamento biológico destas doenças. O objetivo deste estudo é fornecer atualizada do ciclo celilar, carcinogênese, genética do câncer e das diferentes vias bioquímicas envolvidas na gênese das neoplasias astrocitárias.