Application Route and Immune Status of the Host Determine Safety and Oncolytic Activity of Oncolytic Coxsackievirus B3 Variant PD-H.

The coxsackievirus B3 strain PD-0 has been proposed as a new oncolytic virus for the treatment of colorectal carcinoma. Here, we generated a cDNA clone of PD-0 and analyzed the virus PD-H, newly generated from this cDNA, in xenografted and syngenic models of colorectal cancer. Replication and cytotoxic assays revealed that PD-H replicated and lysed colorectal carcinoma cell lines in vitro as well as PD-0. Intratumoral injection of PD-H into subcutaneous DLD-1 tumors in nude mice resulted in strong inhibition of tumor growth and significantly prolonged the survival of the animals, but virus-induced systemic infection was observed in one of the six animals. In a syngenic mouse model of subcutaneously growing Colon-26 tumors, intratumoral administration of PD-H led to a significant reduction of tumor growth, the prolongation of animal survival, the prevention of tumor-induced cachexia, and the elevation of CD3+ and dendritic cells in the tumor microenvironment. No virus-induced side effects were observed. After intraperitoneal application, PD-H induced weak pancreatitis and myocarditis in immunocompetent mice. By equipping the virus with target sites of miR-375, which is specifically expressed in the pancreas, organ infections were prevented. Moreover, employment of this virus in a syngenic mouse model of CT-26 peritoneal carcinomatosis resulted in a significant reduction in tumor growth and an increase in animal survival. The results demonstrate that the immune status of the host, the route of virus application, and the engineering of the virus with target sites of suitable microRNAs are crucial for the use of PD-H as an oncolytic virus.

COVID-19-related cardiac complications from clinical evidences to basic mechanisms: opinion paper of the ESC Working Group on Cellular Biology of the Heart.

The pandemic of coronavirus disease (COVID)-19 is a global threat, causing high mortality, especially in the elderly. The main symptoms and the primary cause of death are related to interstitial pneumonia. Viral entry also into myocardial cells mainly via the angiotensin converting enzyme type 2 (ACE2) receptor and excessive production of pro-inflammatory cytokines, however, also make the heart susceptible to injury. In addition to the immediate damage caused by the acute inflammatory response, the heart may also suffer from long-term consequences of COVID-19, potentially causing a post-pandemic increase in cardiac complications. Although the main cause of cardiac damage in COVID-19 remains coagulopathy with micro- (and to a lesser extent macro-) vascular occlusion, open questions remain about other possible modalities of cardiac dysfunction, such as direct infection of myocardial cells, effects of cytokines storm, and mechanisms related to enhanced coagulopathy. In this opinion paper, we focus on these lesser appreciated possibilities and propose experimental approaches that could provide a more comprehensive understanding of the cellular and molecular bases of cardiac injury in COVID-19 patients. We first discuss approaches to characterize cardiac damage caused by possible direct viral infection of cardiac cells, followed by formulating hypotheses on how to reproduce and investigate the hyperinflammatory and pro-thrombotic conditions observed in the heart of COVID-19 patients using experimental in vitro systems. Finally, we elaborate on strategies to discover novel pathology biomarkers using omics platforms.

Myeloid-Derived Suppressor Cells Restrain Natural Killer Cell Activity in Acute Coxsackievirus B3-Induced Myocarditis.

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis well represent the different outcomes of this inflammatory heart disease. Previously, we found that CVB3-infected A.BY/SnJ mice, susceptible for severe acute and chronic myocarditis, have lower natural killer (NK) cell levels than C57BL/6 mice, with mild acute myocarditis. There is evidence that myeloid-derived suppressor cells (MDSC) may inhibit NK cells, influencing the course of myocarditis. To investigate the MDSC/NK interrelationship in acute myocarditis, we used CVB3-infected A.BY/SnJ mice. Compared to non-infected mice, we found increased cell numbers of MDSC in the spleen and heart of CVB3-infected A.BY/SnJ mice. In parallel, S100A8 and S100A9 were increased in the heart, spleen, and especially in splenic MDSC cells compared to non-infected mice. In vitro experiments provided evidence that MDSC disrupt cytotoxic NK cell function upon co-culturing with MDSC. MDSC-specific depletion by an anti-Ly6G antibody led to a significant reduction in the virus load and injury in hearts of infected animals. The decreased cardiac damage in MDSC-depleted mice was associated with fewer Mac3+ macrophages and CD3+ T lymphocytes and a reduced cardiac expression of S100A8, S100A9, IL-1ß, IL-6, and TNF-α. In conclusion, impairment of functional NK cells by MDSC promotes the development of chronic CVB3 myocarditis in A.BY/SnJ mice.

Empagliflozin improves endothelial and cardiomyocyte function in human heart failure with preserved ejection fraction via reduced pro-inflammatory-oxidative pathways and protein kinase Gα oxidation.

AIMS: Sodium-glucose-cotransporter-2 inhibitors showed favourable cardiovascular outcomes, but the underlying mechanisms are still elusive. This study investigated the mechanisms of empagliflozin in human and murine heart failure with preserved ejection fraction (HFpEF). METHODS AND RESULTS: The acute mechanisms of empagliflozin were investigated in human myocardium from patients with HFpEF and murine ZDF obese rats, which were treated in vivo. As shown with immunoblots and ELISA, empagliflozin significantly suppressed increased levels of ICAM-1, VCAM-1, TNF-α, and IL-6 in human and murine HFpEF myocardium and attenuated pathological oxidative parameters (H2O2, 3-nitrotyrosine, GSH, lipid peroxide) in both cardiomyocyte cytosol and mitochondria in addition to improved endothelial vasorelaxation. In HFpEF, we found higher oxidative stress-dependent activation of eNOS leading to PKGIα oxidation. Interestingly, immunofluorescence imaging and electron microscopy revealed that oxidized PKG1α in HFpEF appeared as dimers/polymers localized to the outer-membrane of the cardiomyocyte. Empagliflozin reduced oxidative stress/eNOS-dependent PKGIα oxidation and polymerization resulting in a higher fraction of PKGIα monomers, which translocated back to the cytosol. Consequently, diminished NO levels, sGC activity, cGMP concentration, and PKGIα activity in HFpEF increased upon empagliflozin leading to improved phosphorylation of myofilament proteins. In skinned HFpEF cardiomyocytes, empagliflozin improved cardiomyocyte stiffness in an anti-oxidative/PKGIα-dependent manner. Monovariate linear regression analysis confirmed the correlation of oxidative stress and PKGIα polymerization with increased cardiomyocyte stiffness and diastolic dysfunction of the HFpEF patients. CONCLUSION: Empagliflozin reduces inflammatory and oxidative stress in HFpEF and thereby improves the NO-sGC-cGMP-cascade and PKGIα activity via reduced PKGIα oxidation and polymerization leading to less pathological cardiomyocyte stiffness.

Immune attributes of cardiac-derived adherent proliferating (CAP) cells in cardiac therapy.

Cardiac-directed cell therapies show potential to reduce mortality and morbidity in heart disease. However, high functional efficacy should be complimented with low immunogenicity, in particular if allogeneic cell sources are applied. Therefore, we aimed to examine cardiac-derived adherent proliferating (CAP) cells with respect to their immunogenicity and immune modulatory features in vitro. Human CAP cells were isolated from cardiac biopsies and screened in a CFSE-based proliferation assay in co-cultures with phytohaemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBMCs) or mixed lymphocyte cultures (MLCs) to assess their potential to induce immune cell proliferation or activation by flow cytometry. Moreover, levels of pro- and anti-inflammatory cytokines in supernatants of co-cultures were analysed. The capacity of CAP cells to induce the generation of regulatory T cells (Tregs) was determined by flow cytometric measurement of FoxP3 expression. CAP cells of different donors (n = 5) showed low immunogenicity in co-cultures with human allogeneic PBMCs. In addition, they induced no change in the normal alloantigen-driven immune responsiveness in MLCs. However, CAP cells significantly reduced the induction of immune cell proliferation in PBMCs cultures stimulated with the polyclonal trigger PHA. Adding CAP cells into MLCs or PHA-stimulated cultures resulted in significantly reduced levels of TNFα or IFNγ, respectively, compared to controls without CAP cells. At early time points (day 2), interaction of CAP cells with PBMCs resulted in elevated proportions of FoxP3+ CD4+ CD25(high+) cells. The results indicate that CAP cells have low immunogenicity and could be advantageous in cardiac repair by reducing inflammatory cytokines and inducing regulatory T cells.

Cardiac function and remodeling is attenuated in transgenic rats expressing the human kallikrein-1 gene after myocardial infarction.

Bradykinin coronary outflow, left ventricular performance and left ventricular dimensions of transgenic rats harboring the human tissue kallikrein-1 gene TGR(hKLK1) were investigated under basal and ischemic conditions. Bradykinin content in the coronary outflow of buffer-perfused, isolated hearts of controls and TGR(hKLK1) was measured by specific radioimmunoassay before and after global ischemia. Left ventricular function and left ventricular dimensions were determined in vivo using a tip catheter and echocardiography 6 days and 3 weeks after induction of myocardial infarction. Left ventricular type I collagen mRNA expression was analyzed by RNase protection assay. Compared to controls, basal bradykinin outflow was 3.5 fold increased in TGR(hKLK1). Ischemia induced an increase of bradykinin coronary outflow in controls but did not induce a further increase in TGR(hKLK1). However, despite similar unchanged infarction sizes, left ventricular function and remodeling improved in TGR(hKLK1) after myocardial infarction, indicated by an increase in left ventricular pressure (+34%; P<0.05), contractility (dp/dt max. +25%; P<0.05), and in ejection fraction (+20%; P<0.05) as well as by a reduction in left ventricular enddiastolic pressure (-49%, P<0.05), left ventricular enddiastolic diameter (-20%, P<0.05), and collagen mRNA expression (-15%, P<0.05) compared to controls. A chronically activated transgenic kallikrein kinin system with expression of human kallikrein-1 gene counteracts the progression of left ventricular contractile dysfunction after experimental myocardial infarction. Further studies have to show whether these results can be caused by other therapeutically options. Long acting bradykinin receptor agonists might be an alternative option to improve ischemic heart disease.