Andrea Paccone

Article: Retraction of: “Sacubitril/valsartan improves left ventricular longitudinal deformation in heart failure patients with reduced ejection fraction” - Minerva Cardiology and Angiology 2023 October;71(5):609 - Minerva Medica - Journals

Immune checkpoint inhibitors (ICIs) have significantly changed the oncology clinic in recent years, improving survival expectations in cancer patients. ICI therapy have a broad spectrum of side effects from endocrinopathies to cardiovascular diseases. In this study, pro–inflammatory and pro–fibrotic effects of short–term ICIs therapy in preclinical models were analyzed. Firstly, in a human in vitro model, human cardiomyocytes co–cultured with hPBMC were exposed to ICIs (with CTLA–4 or PD–1 blocking agents, at 200 nM) for 72h. After treatment, production of DAMPs and 12 cytokines were analyzed in the supernatant through colorimetric and enzymatic assays. C57/Bl6 mice were treated with CTLA–4 or PD–1 blocking agents (15 mg/kg) for 10 days. Before (T0), after three days (T3) and after treatments (T10), ejection fraction, fractional shortening, radial and longitudinal strain were calculated by using bidimensional echocardiography (Vevo 2100,Fujfilm). Fibrosis, necrosis, hypertrophy and vascular NF–kB expression were analyzed through Immunohistochemistry. Myocardial expression of DAMPs (S100– Calgranulin, Fibronectin and Galectine–3), MyD88, NLRP3 and twelve cytokines have been analyzed. Systemic levels of SDF–1, IL–1β and IL–6 were analyzed before, during and after ICIs therapy. Radial and longitudinal strain were decreased after 10 days of ICIs therapy. Histological analysis of NF–kB expression shows that short–term anti–CTLA–4 or anti–PD–1 treatment increased vascular and myocardial inflammation. No myocardial hypertrophy was seen with the exception of the pembrolizumab group. Myocardial fibrosis and expression of galectin–3, pro–collagen 1–α and MMP–9 were increased after treatment with all ICIs. Both anti–CTLA–4 or anti–PD–1 treatments increased the expression of DAMPs, NLRP3 inflammasome and MyD88 and induced both in vitro and in vivo the secretion of IL–1β, TNF–α and IL–6. Systemic levels of SDF–1, IL–1β and IL–6 were increased during and after treatment with ICIs. Short therapy with PD–1 and CTLA–4 blocking agents increases vascular expression of NF–kB, systemic SDF–1, IL–1β, IL–6 levels and myocardial NLRP3, MyD88 and DAMPs expression in preclinical models. A pro–inflammatory cytokine storm was induced in myocardial tissues and in cultured cardiac cells after ICIs therapy.

Anthracyclines are an effective and widely used chemotherapy agent in the treatment of multiple solid organ tumors and hematologic malignancies. The use of anthracyclines as a standard cancer therapy is limited by the potential for the development of cardiac dysfunction, arrhythmias, and clinical heart failure. In recent five years, it was demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9), a lipid metabolism–related protein, is a key orchestrator of immune infiltration in myocardial and cancer tissues and could regulate cardiac fibrosis and inflammation. PCSK9 is a protein with key roles in hepatic low density lipoprotein (LDL) homeostasis. PCSK9 systemic levels are associated to HOMA score and high insulin levels. Dapagliflozin exerts systemic ant–inflammatory properties and cardioprotective effects in diabetic and non–diabetic patients. Purpose: We hypothesized that Dapagliflozin, administered during doxorubicin, could reduce PCSK9 systemic levels in preclinical models. Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), DAPA at 12 mg/kg (DAPA, n=6) or doxorubicin combined to DAPA (DOXO–DAPA, n=6). After treatments, plasma levels of PCSK9, IL–1β and CRP were analyzed through selective anti–mouse ELISA methods. Myocardial and liver expression of NLRP3–inflammasome and IL–1β were analyzed through ELISA method in tissue lysates after treatments. DAPA associated to DOXO reduces significantly systemic levels of PCSK9 ( –37,5% vs DOXO group, p<0,001). IL–1β and CRP levels were also reduced ( –47,3 and – 28,5 %, respectively; p<0.05 for both). Myocardial and liver IL–1β and NLRP3 inflammasome expression were also reduced in DAPA/DOXO group vs DOXO and control, indicated beneficial metabolic and anti–inflammatory effects of SGLT2i. DAPA has been shown to reduce systemic levels of PCSK9 in preclinical models of short–term DOXO cardiotoxicity. To the best of our knowledge, this is the first evidence of SGLT–2/PCSK9 crass–talk in cardioncology therefore the overall picture of the study open a new window on the beneficial properties of DAPA against anthracyclines side effects.

Background: Anthracyclines are essential adjuvant therapies for a variety of cancers, particularly breast, and gastric and esophageal cancers. Whilst prolonging cancer–related survival, these agents can in–duce drug–related cardiotoxicity. Spirulina, Reishi (Ganoderma Lucidum) and Moringa are three nutaceuticals with anti–inflammatory effects that are currently used in cancer patients to improve quality of life and fatigue. Methods: Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with short–term doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), Singo at 12 mg/kg (Singo, n=6) or doxorubicin combined to Singo (DOXO–Singo, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100, Fujifilm). Myocardial expression of NLRP3, DAMPs (galectine 3 and calgranulin S100) and 13 cytokines (IL–1α, IL–1β, IL–2, IL–4, IL–6, IL–10, IL–12, IL17–α, IL–18, IFN–γ, TNF–α, G–CSF, and GM–CSF) were quantified through selective mouse ELISA methods. Myocadial fibrosis, necrosis and hypertrophy were analyzed through Immunohistochemistry (IHC). Human cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin (200 nM) alone or in combination to Singo ( at 10, 25 and 50 µg/ml) for 24 and 48h. Cell viability studies were performed through MTS assay. Expression of NLRP3 and p65/NF–kB) were made through selective ELISA methods. Results: In preclinical models of short–term DOXO cardiotoxicity, Singo improved significantly EF and FS and prevented the reduction of radial and longitudinal strain after 10 days of treatment with DOXO. A reduced expression of myocardial NLRP3 and NF–kB levels in cardiac tissues were seen in DOXO–Singo group compared to DOXO mice (p<0.05). Myocardial expression of pro–inflammatory cytokines were significantly reduced after treatment with Singo indicating anti–inflammatory properties. Myocardial levels of Cal–granulin S100 and galectine–3 were strongly enhanced in DOXO group; on the other hand their expression were reduced in Singo–DOXO group vs DOXO (p<0.05). Immunohistochemistry analysis indicates that Singo reduces fibrosis and hypertrophy in myocardial tissue of mice during exposure to DOXO. Conclusion: Singo is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis. Cardioprotective properties of Singo are mediated by the reduction of lipid peroxidation products and expression of NLRP3–NF–kB –cytokine pathways.

Background: Several strategies based on immune checkpoint inhibitors (ICIs) have been developed or are under investigation for cancer therapy, opening to advantages in cancer outcomes. However, several ICIs-induced side effects emerged in these patients, especially a rare but clinically significant cardiotoxicity with high rate of mortality. Purpose: We analyzed the differential vasculo and cardiotoxicity of Pembrolizumab, Nivolumab and Ipilimumab in preclinical models highlighting on the molecular pathways involved. Methods: C57 female mice were treated with Ipilimumab, Pembrolizumab or Nivolumab (15 mg/kg) through intraperitoneal injection for 10 days. pAMPK expression and vascular p65/NF-kB was analyzed through Immunohistochemistry. Cardiac levels of IL-1α, IL-1β, IL-2, IL-6 and IL-10 were quantified through ELISA method Results: In preclinical models, all ICIs increased p65/NF-kB expression in vascular endothelial cells of mice compared to saline group. IHC analysis indicates that ICIs reduced pAMPK expression in myocardial cells. Notably, tissue levels of IL-1α, IL-1β, IL-2, IL-6 were strongly increased in ICIs group vs saline (p<0.05 vs saline). On the other hand, levels of the anti-inflammatory cytokine IL-10 were strongly reduced (overall reduction of 43.3–54.5% vs saline) Conclusion: In preclinical models, a short treatment with ICIs is sufficient to confer a pro-inflammatory phenotype in the heart of mice. Furthermore, reductions in pAMPK indicate the inhibition of mitochondrial metabolism after ICIs. Furthermore ICIs induce chemokines involved in fibrosis, myocarditis and vascultitis. These results could indicate new therapeutic candidates aimed to prevent myocardial and vascular damages in cancer patients treated with ICIs. Funding Acknowledgement: Type of funding sources: Public Institution(s). Main funding source(s): Ricerca Corrente, Ministero della Salute