LOGAN-CV: A Prospective Study of a Multifaceted Intervention Targeting United States Clinicians to Improve Guideline-Based Management of Lipid-Lowering Therapy.

INTRODUCTION: The 2018 American Heart Association (AHA)/American College of Cardiology (ACC)/Multisociety blood cholesterol guidelines recommend clinicians consider adding non-statin therapy for patients with very high-risk (VHR) atherosclerotic cardiovascular disease (ASCVD) and low-density lipoprotein cholesterol (LDL-C) ≥ 70 mg/dl while receiving maximally tolerated statins. However, according to a recent study, only 17.1% of patients with established ASCVD received appropriate lipid-lowering therapy (LLT) intensification. Here, we describe the design of a prospective, 12-month study (LOGAN-CV) evaluating a multifaceted site-level intervention to enhance clinicians' adherence to guidelines to improve LDL-C levels for patients with VHR ASCVD. METHODS: Clinicians from up to ten research sites are eligible if they care for adult patients with ASCVD. Interventions include educational modules, a cloud-based performance platform providing clinicians a tailored summary of their LDL-C management performance, newsletters, periodic peer-to-peer calls, and pre- and post-intervention surveys evaluating knowledge, attitudes, and beliefs around LDL-C management, with additional interventions for clinicians demonstrating a lower readiness to make treatment decisions based on guideline recommendations. Patients with VHR ASCVD, defined as having recent myocardial infarction and LDL-C ≥ 70 mg/dl despite statin treatment, will be included in the study. Patient data will be collected from electronic medical records from baseline (clinician enrollment) through the 12-month intervention. The study started in October 2022, with anticipated completion in March 2024. PLANNED OUTCOMES: The change in proportion of patients with LDL-C < 70 mg/dl achieved at any time during the 12-month intervention (primary); LLT intensification, changes in guideline-aligned LDL-C testing and LLT titration over 12 months, and change in overall clinicians' knowledge, attitudes, and beliefs are key outcomes of interest. The LOGAN-CV study addresses a critical unmet need in LDL-C control in patients with VHR ASCVD and evaluates the effect of a multifaceted intervention targeting clinicians to improve their adherence to guidelines and consequently improve clinical outcomes for patients.

Homotypic Entosis as a Potential Novel Diagnostic Marker in Breast Cancer.

Homotypic entotic figures, which are a form of "cell-in-cell" structures, are considered a potential novel independent prognostic marker in various cancers. Nevertheless, the knowledge concerning the biological role of this phenomenon is still unclear. Since breast cancer cells are remarkably entosis-competent, we aimed to investigate and compare the frequency of entoses in a primary breast tumor and in its lymph node metastasis. Moreover, as there are limited data on defined molecular markers of entosis, we investigated entosis in correlation with classical breast cancer biomarkers used in routine pathomorphological diagnostics (HER2, ER, PR, and Ki67). In the study, a cohort of entosis-positive breast cancer samples paired into primary lesions and lymph node metastases was used. The inclusion criteria were a diagnosis of NOS cancer, lymph node metastases, the presence of entotic figures in the primary lesion, and/or lymph node metastases. In a selected, double-negative, HER2-positive NOS breast cancer case, entoses were characterized by a correlation between an epithelial-mesenchymal transition and proliferation markers. We observed that in the investigated cohort entotic figures were positively correlated with Ki67 and HER2, but not with ER or PR markers. Moreover, for the first time, we identified Ki67-positive mitotic inner entotic cells in clinical carcinoma samples. Our study performed on primary and secondary breast cancer specimens indicated that entotic figures, when examined by routine HE histological staining, present potential diagnostic value, since they correlate with two classical prognostic factors of breast cancer.

Hepatic Sensing Loop Regulates PCSK9 Secretion in Response to Inhibitory Antibodies.

BACKGROUND: Monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9i) lower LDL-C by up to 60% and increase plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) levels by 10-fold. OBJECTIVES: The authors studied the reasons behind the robust increase in plasma PCSK9 levels by testing the hypothesis that mechanisms beyond clearance via the low-density lipoprotein receptor (LDLR) contribute to the regulation of cholesterol homeostasis. METHODS: In clinical cohorts, animal models, and cell-based studies, we measured kinetic changes in PCSK9 production and clearance in response to PCSK9i. RESULTS: In a patient cohort receiving PCSK9i therapy, plasma PCSK9 levels rose 11-fold during the first 3 months and then plateaued for 15 months. In a cohort of healthy volunteers, a single injection of PCSK9i increased plasma PCSK9 levels within 12 hours; the rise continued for 9 days until it plateaued at 10-fold above baseline. We recapitulated the rapid rise in PCSK9 levels in a mouse model, but only in the presence of LDLR. In vivo turnover and in vitro pulse-chase studies identified 2 mechanisms contributing to the rapid increase in plasma PCSK9 levels in response to PCSK9i: 1) the expected delayed clearance of the antibody-bound PCSK9; and 2) the unexpected post-translational increase in PCSK9 secretion. CONCLUSIONS: PCSK9 re-entry to the liver via LDLR triggers a sensing loop regulating PCSK9 secretion. PCSK9i therapy enhances the secretion of PCSK9, an effect that contributes to the increased plasma PCSK9 levels in treated subjects.

Classification of Cell-in-Cell Structures: Different Phenomena with Similar Appearance.

A phenomenon known for over 100 years named "cell-in-cell" (CIC) is now undergoing its renaissance, mostly due to modern cell visualization techniques. It is no longer an esoteric process studied by a few cell biologists, as there is increasing evidence that CICs may have prognostic and diagnostic value for cancer patients. There are many unresolved questions stemming from the difficulties in studying CICs and the limitations of current molecular techniques. CIC formation involves a dynamic interaction between an outer or engulfing cell and an inner or engulfed cell, which can be of the same (homotypic) or different kind (heterotypic). Either one of those cells appears to be able to initiate this process, which involves signaling through cell-cell adhesion, followed by cytoskeleton activation, leading to the deformation of the cellular membrane and movements of both cells that subsequently result in CICs. This review focuses on the distinction of five known forms of CIC (cell cannibalism, phagoptosis, enclysis, entosis, and emperipolesis), their unique features, characteristics, and underlying molecular mechanisms.

Novel podophyllotoxin and benzothiazole derivative induces transitional morphological and functional changes in HaCaT cells.

Podophyllotoxin (PPT) is an antimitotic drug used topically in the treatment of anogenital warts. Due to its toxicity it cannot be administered systemically as an anticancer agent. However, modified PPT derivatives such as etoposide and teniposide are used clinically as systemic agents. Thus, we invented novel PPT derivative KL3 that was synthesized by photocyclization. Earlier we have shown that KL3 has an anticancer effect in various cell lines. Here we compared the toxicity of KL3 vs PPT on non-cancerous normal human keratinocytes (HaCaT) and peripheral blood mononuclear cells (PBMC) showing that KL3 is less toxic than PPT to non-cancerous cells. At concentrations that neither induced cell death, nor affected cell cycle, KL3 in HaCaT cells evoked transient ultrastructural features of ER stress, swelling of mitochondria and elongation of cytoplasmic processes. Those changes partially reversed with prolonged incubation while features of autophagy were induced. PPT in equivalent concentrations induced HaCaT cell death by cell cycle arrest, intrinsic apoptosis and finally disintegration of cell membranes followed by secondary necrosis. In conclusion, we show that the KL3 derivative of PPT in contrast to PPT allows repair of normal keratinocytes and triggers mechanisms that restore non-tumor cell homeostasis.

Entosis: From Cell Biology to Clinical Cancer Pathology.

Entosis is a phenomenon, in which one cell enters a second one. New clinico-histopathological studies of entosis prompted us to summarize its significance in cancer. It appears that entosis might be a novel, independent prognostic predictor factor in cancer histopathology. We briefly discuss the biological basis of entosis, followed by a summary of published clinico-histopathological studies on entosis significance in cancer prognosis. The correlation of entosis with cancer prognosis in head and neck squamous cell carcinoma, anal carcinoma, lung adenocarcinoma, pancreatic ductal carcinoma and breast ductal carcinoma, is shown. Numerous entotic figures are associated with a more malignant cancer phenotype and poor prognosis in many cancers. We also showed that some anticancer drugs could induce entosis in cell culture, even as an escape mechanism. Thus, entosis is likely beneficial for survival of malignant cells, i.e., an entotic cell can hide from unfavourable factors in another cell and subsequently leave the host cell remaining intact, leading to failure in therapy or cancer recurrence. Finally, we highlight the potential relationship of cell adhesion with entosis in vitro, based on the model of the BxPc3 cells cultured in full adhesive conditions, comparing them to a commonly used MCF7 semiadhesive model of entosis.

Emerging lipid lowering agents targeting LDL cholesterol.

Atherosclerotic cardiovascular disease (ASCVD) is the main cause of morbidity and mortality in the US. ASCVD is caused by elevated levels of ApoB lipoproteins, which over many years penetrate the arterial subendothelial space leading to plaque growth and eventually rupture causing clinical symptoms. ApoB lipoprotein levels are approximated in clinical practice by LDL-C measurement. LDL-C lowering agents (statins, ezetimibe, and PCSK9 inhibitors) reduce cardiovascular risk in primary and secondary prevention proportionally to LDL-C reduction (23% per 1 mmol/L of LDL). However, for a variety of reasons, many patients do not achieve their recommended LDL-C levels using currently available therapies. This has prompted the development of new LDL-C lowering drugs in the hope to reduce cardiovascular risk, such as bempedoic acid, inclisiran, gemcabene, and evinacumab. Drugs targeting other lipids (triglycerides, HDL-C, lipoprotein (a)), intravascular inflammation or acting by other mechanisms also have a role in atherosclerosis prevention, however, they will not be covered in this review. ABBREVIATIONS: ACLY: (ATP-citrate lyase); ANGPTL: (angiopoietin-like protein 3); ASCVD: (atherosclerotic cardiovascular disease); BPA: (bempedoic acid); CETP (cholesteryl ester transfer protein); CV: (cardiovascular); CVD: (cardiovascular diseases); FH (familial hypercholesterolemia); HMGCR (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase); HoFH (homozygous FH); LDL-C: (low density lipoprotein cholesterol); LDL-P: (low density lipoprotein particle); LDLr: (low density lipoprotein receptor); NPC1L1: (Niemann-Pick C1-like 1 protein); PCSK9: (proprotein convertase subtilisin/kexin type 9); SREBP-2: (sterol regulatory element binding protein 2).

Mechanisms and Evidence for Heart Failure Benefits from SGLT2 Inhibitors.

PURPOSE OF REVIEW: To review the clinical trial data and underlying mechanistic principles in support of the robust cardiovascular (CV) benefits, in particular, heart failure (HF) outcomes association with sodium-glucose co-transporter-2 (SGLT2) inhibitors. RECENT FINDINGS: Several large CV outcome trials in patients with type 2 diabetes mellitus (T2DM) and with either established atherosclerotic CV disease (ASCVD) or at high risk for ASCVD reveal that SGLT2 inhibitors cause reductions in CV and HF endpoints. The reduction in ASCVD appears to be confined to those with established ASCVD on the order of ≈ 14%, as does the mortality benefit-all-cause and CV-related. However, hospitalization for HF are reduced by ≈ 33% and occur regardless of baseline patient characteristics. The unprecedented HF outcomes are theorized to occur via several possible mechanisms and include optimization of conventional ASCVD risk factors, improvement in hemodynamics, prevention of cardiac and renal remodeling, inhibition of hormone dysregulation, use of more efficient metabolic substrates, ion channel inhibition, anti-inflammatory effects, and anti-oxidant effects. Recent evidence has unveiled the irrefutable data that SGLT2 inhibitors reduce CV events in patients with T2DM, with a profound effect on reductions in hospitalization for HF. Though several mechanisms conveying this benefit are suggested, most are based in limited data requiring further validation. Nonetheless, the arrival of SGLT2 inhibitors has ushered in a new era of CV risk reductions therapies.

Translating AHA/ACC cholesterol guidelines into meaningful risk reduction.

The new recommendations detail refined, personalized lipid management and emphasize multiple levels of evidence. The result? Care is more complex but patients might benefit more.

Application of PCSK9 Inhibitors in Practice.

PCSK9i (protein convertase subtilisin/kexin type 9 inhibitors) are set to revolutionize the treatment of hypercholesterolemia in the management of atherosclerotic risk, but numerous reports have detailed unprecedented barriers to access for these drugs. To overcome these challenges, our group created a model to facilitate provision of this new therapy for patients who qualify according to Food and Drug Administration criteria. This report details the real-world follow-up experience of PCSK9i use in a large patient cohort structured to ensure rigor in data collection, analysis, and interpretation. The 271 patients approved and actively followed in our PCSK9i clinic between July 2015 and August 2018 represent a 97% approval rate from insurance, with 28% of prescriptions requiring at least one appeal. Over 50% of patients were statin intolerant. On average, there was a median lapse of 15 days between initial visit and insurance approval. PCSK9i therapy was affordable for most patients, with an average monthly out-of-pocket expense of $58.05 (median $0). Only 2.3% of patients were unable to initiate or continue therapy because of cost. Reductions from baseline in LDL (low-density lipoprotein) cholesterol and Lp(a) (lipoprotein [a])were comparable to published reports with median reductions of 60% and 23% at 1 year, respectively. PCSK9i therapy was well-tolerated overall, though 9% of patients reported adverse events, and 5% of patients discontinued due mostly to musculoskeletal and flu-like symptoms. Our practice model demonstrates that PCSK9i therapy can be accessed easily and affordably for the majority of eligible patients, resulting in dramatic improvement in lipid profile results. Moreover, our registry data suggest that results from the prospective clinical trials of PCSK9i on LDL and Lp(a) reduction and on tolerability are applicable to a real-world cohort.

Co-occurrence of heterozygous CREB3L3 and APOA5 nonsense variants and polygenic risk in a patient with severe hypertriglyceridemia exacerbated by estrogen administration.

We describe a case of a 36-year-old woman with severe hypertriglyceridemia likely caused by double heterozygosity of a known pathogenic APOA5 nonsense variant (p.Q275X) and a novel CREB3L3 nonsense variant (p.C296X) on a background of very strong polygenic susceptibility. Her clinical course worsened with development of eruptive xanthomata after oral administration of 2 mg estradiol twice daily for 2 weeks as part of a medical protocol for intrauterine embryo transfer following in vitro fertilization. Her triglyceride levels decreased to baseline and xanthomata resolved without treatment after discontinuation of hormonal therapy, which also resulted in termination of pregnancy. Before undergoing a second embryo transfer using her natural cycle and no exogenous hormones, the patient started combination therapy with eicosapentaenoic acid ethyl ester and gemfibrozil, leading to an ∼80% decrease in triglyceride levels. She continued treatment throughout pregnancy, which progressed to term with the delivery of healthy twins.

Mucosal delivery systems of antihypertensive drugs: A practical approach in general practice.

Patients who are unable to receive oral medication (p.o.) are a major problem in outpatient settings, especially in home health care systems. Mucosal administration of drugs offers an alternative to the oral route, especially when the parenteral mode cannot be used. There are three main pathways of mucosal administration: sublingual/buccal, intranasal and rectal. We discuss the possibility of mucosal delivery of antihypertensive drugs. Perindopril arginine and Amlodipine besylate are registered in the EU as orodispersible tablets for oromucosal delivery, however, they are not available in all countries. For this reason, we describe other drugs suitable for mucosal delivery: Captopril and Nitrendipine in the sublingual system and Metoprolol tartrate, Propranolol and Furosemide by the transrectal route. Based on the published data and common clinical practice we discuss the use of mucosal delivery systems of all these antihypertensive drugs with special attention to their pharmacokinetics. We illustrate this mini-review with a case report of the prolonged-term use of mucosal delivery of sublingual Captopril and Nitrendipine combined with rectal Metoprolol tartrate and Furosemide in a patient with severe hypertension unable to receive medication p.o. This is also a report on the first human use of Furosemide-containing suppositories as well as prolonged-term transmucosal administration of these four drugs, describing a practical approach leading to successful control of severe hypertension with four antihypertensive drugs delivered via the mucosal route. The treatment was effective and without side effects; however, the long-term safety and efficacy of such therapy must be confirmed by randomized clinical trials.

Incorporation of PCSK9 inhibitors into prevention of atherosclerotic cardiovascular disease.

Primary and secondary prevention of atherosclerotic cardiovascular disease (ASCVD) has become recently more complex than ever, leaving the clinicians perplexed with outdated guidelines and emerging evidence about new LDL-C lowering therapies. 2013 American College of Cardiology (ACC)/American Heart Association (AHA) guidelines have focused on high intensity statin therapy for specific groups of patients, while abandoning long established LDL-C goals, a strategy which no longer seems valid. PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors have emerged as the add-on therapy on top of statins and/or ezetimibe for the treatment of hypercholesterolemia and ASCVD prevention. In several clinical trials, PCSK9 inhibitors have demonstrated their safety and robust LDL-C-lowering power. One completed cardiovascular (CV) outcomes trial (FOURIER; Further Cardiovascular Outcomes Research with PCSK9 Inhibitions in Subjects with Elevated Risk) has demonstrated that PCSK9 inhibition reduces rates of CV death as well as non-fatal stroke and MI, while another major CV outcome trial is under way (ODYSSEY-OUTCOMES). Several trials studying CV benefits of novel LDL-C-lowering therapies are also being conducted. Prompt revision of ACC/AHA guidelines is necessary. In the meantime, physicians need to use clinical judgment integrating the most recent evidence into their practice.

Discordant response of low-density lipoprotein cholesterol and lipoprotein(a) levels to monoclonal antibodies targeting proprotein convertase subtilisin/kexin type 9.

BACKGROUND: Clinical trials testing proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) have demonstrated an unanticipated but significant lipoprotein (a) (Lp(a))-lowering effect, on the order of 25% to 30%. Although the 50% to 60% reduction in low-density lipoprotein (LDL)-cholesterol (LDL-C) achieved by PCSK9i is mediated through its effect on LDL receptor (LDLR) preservation, the mechanism for Lp(a) lowering is unknown. OBJECTIVE: We sought to characterize the degree of concordance between LDL-C and Lp(a) lowering because of PCSK9i in a standard of care patient cohort. METHODS: Participants were selected from our Center for Preventive Cardiology, an outpatient referral center in a tertiary academic medical center. Subjects were included in this study if they had (1) at least 1 measurement of LDL-C and Lp(a) before and after initiation of the PCSK9i; (2) baseline Lp(a) > 10 mg/dL; and (3) continued adherence to PCSK9i therapy. They were excluded if (1) they were undergoing LDL apheresis; (2) pre- or post-PCSK9i LDL-C or Lp(a) laboratory values were censored; or (3) subjects discontinued other lipid-modifying therapies. In total, 103 subjects were identified as taking a PCSK9i and 26 met all inclusion and exclusion criteria. Concordant response to therapy was defined as an LDL-C reduction >35% and an Lp(a) reduction >10%. RESULTS: The cohort consisted of 26 subjects (15 females, 11 males, mean age 63 ± 12 years). Baseline mean LDL-C and median Lp(a) levels were 167.4 ± 72 mg/dL and 81 mg/dL (interquartile range 38-136 mg/dL), respectively. The average percent reductions in LDL-C and Lp(a) were 52.8% (47.0-58.6) and 20.2% (12.2-28.1). The correlation between %LDL and %Lp(a) reduction was moderate, with a Spearman's correlation of 0.56 (P < .01). All subjects except for 1 had a protocol-appropriate LDL-C response to therapy. However, only 16 of the 26 (62%; 95% confidence interval 41%-82%) subjects had a protocol-concordant Lp(a) response. Although some subjects demonstrated negligible Lp(a) reduction associated with PCSK9i, there were some whose Lp(a) decreased as much as 60%. CONCLUSIONS: In this standard-of-care setting, we demonstrate moderate correlation but large discordance (∼40%) in these 2 lipid fractions in response to PCSK9i. The results suggest that pathways beyond the LDLR are responsible for Lp(a) lowering and indicate that PCSK9i have the potential to significantly lower Lp(a) in select patients, although confirmation in larger multicenter studies is required.

Modulation of adipocyte differentiation by omega-3 polyunsaturated fatty acids involves the ubiquitin-proteasome system.

We have evaluated the effects of three different omega-3 polyunsaturated fatty acids (ω-3 PUFAs) ­ docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) on fat accumulation and expression of adipogenic and inflammatory markers using both 3T3-L1 pre-adipocytes and differentiated 3T3-L1 adipocytes. Our results indicate that ω-3 PUFAs induce the degradation of fatty acid synthase through the ubiquitin-proteasome system, which is likely to have beneficial metabolic effect on adipose cells. Omega-3 PUFAs also increase overall levels of polyubiquitinated proteins, at least in part through decreasing the expression of proteasome subunits. Moreover, adipocytes are resistant to proteasome inhibition, which induces adipophilin while decreasing perilipin expression. On the other hand, ω-3 PUFAs decrease expression of SREBP1 while inducing expression of adipophilin and GLUT4. Moreover, all three ω-3 PUFAs appear to induce tumour necrosis factor-α without affecting NFκB levels. All three ω-3 PUFAs appear to have overall similar effects. Further research is needed to elucidate their mechanism of action.