BMP2 gene transfer induces pericardial effusion and inflammatory response in the ischemic porcine myocardium

Pro-angiogenic gene therapy is being developed to treat coronary artery disease (CAD). We recently showed that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor-A synergistically regulate endothelial cell sprouting in vitro. BMP2 was also shown to induce endocardial angiogenesis in neonatal mice post-myocardial infarction. In this study, we investigated the potential of BMP2 gene transfer to improve cardiomyocyte function and neovessel formation in a pig chronic myocardial infarction model. Ischemia was induced in domestic pigs by placing a bottleneck stent in the proximal part of the left anterior descending artery 14 days before gene transfer. Intramyocardial gene transfers with adenovirus vectors (1 × 1012 viral particles/pig) containing either human BMP2 (AdBMP2) or beta-galactosidase (AdLacZ) control gene were performed using a needle injection catheter. BMP2 transgene expression in the myocardium was detected with immunofluorescence staining in the gene transfer area 6 days after AdBMP2 administration. BMP2 gene transfer did not induce angiogenesis or cardiomyocyte proliferation in the ischemic pig myocardium as determined by the quantitations of CD31 or Ki-67 stainings, respectively. Accordingly, no changes in heart contractility were detected in left ventricular ejection fraction and strain measurements. However, BMP2 gene transfer induced pericardial effusion (AdBMP2: 9.41 ± 3.17 mm; AdLacZ: 3.07 ± 1.33 mm) that was measured by echocardiography. Furthermore, an increase in the number of immune cells and CD3+ T cells was found in the BMP2 gene transfer area. No changes were detected in the clinical chemistry analysis of pig serum or histology of the major organs, implicating that the gene transfer did not induce general toxicity, myocardial injury, or off-target effects. Finally, the levels of fibrosis and cardiomyocyte apoptosis detected by Sirius red or caspase 3 stainings, respectively, remained unaltered between the groups. Our results demonstrate that BMP2 gene transfer causes inflammatory changes and pericardial effusion in the adult ischemic myocardium, which thus does not support its therapeutic use in chronic CAD

The Gag Cleavage Product, p12, is a Functional Constituent of the Murine Leukemia Virus Pre-Integration Complex

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration

Vascular Remodeling in Health and Disease

The term vascular remodeling is commonly used to define the structural changes in blood vessel geometry that occur in response to long-term physiologic alterations in blood flow or in response to vessel wall injury brought about by trauma or underlying cardiovascular diseases.1, 2, 3, 4 The process of remodeling, which begins as an adaptive response to long-term hemodynamic alterations such as elevated shear stress or increased intravascular pressure, may eventually become maladaptive, leading to impaired vascular function. The vascular endothelium, owing to its location lining the lumen of blood vessels, plays a pivotal role in regulation of all aspects of vascular function and homeostasis.5 Thus, not surprisingly, endothelial dysfunction has been recognized as the harbinger of all major cardiovascular diseases such as hypertension, atherosclerosis, and diabetes.6, 7, 8 The endothelium elaborates a variety of substances that influence vascular tone and protect the vessel wall against inflammatory cell adhesion, thrombus formation, and vascular cell proliferation.8, 9, 10 Among the primary biologic mediators emanating from the endothelium is nitric oxide (NO) and the arachidonic acid metabolite prostacyclin [prostaglandin I2 (PGI2)], which exert powerful vasodilatory, antiadhesive, and antiproliferative effects in the vessel wall

Unveiling the role of bovine CYP1A1 and CYP3A28 in AFB1 metabolism: molecular docking and CRISPR/Cas9-mediated genetic knockout in BFH12 cells

Introduction: Cytochromes P450 (CYPs) represent a multigene family of monooxygenase proteins involved in the biotransformation of endogenous compounds and xenobiotics. Aflatoxin B1 (AFB1) is a mycotoxin occurring in several food and feed commodities. In human liver, AFB1 is metabolized by CYP1A and CYP3A isoforms in different carcinogenic and toxic metabolites, such as AFB1-exo-8,9-epoxide (AFBO) and AFM1, and relatively nontoxic derivatives (AFQ1 and AFP1) (1). In cattle, CYP1A1 and CYP3A28 seem crucial for AFB1 metabolism, but their specific role has not been thoroughly investigated yet (2). In this study, AFB1 molecular docking into the abovementioned bovine CYPs was performed. Moreover, the generation of CYP1A1 and CYP3A28 knockout (KO) BFH12 cell lines was exploited to elucidate the role of these two CYPs in AFB1 metabolism. Materials and Methods: Homology modelling using human CYP1A1 (PDB:4I8V) and CYP3A4 (PDB:5TE8) in complex with alpha-naphthoflavone or midazolam, respectively, was preliminarily conducted to build bovine CYP1A1 and CYP3A28 models; then, docking of AFB1 with Glide was performed into these models (Schrödinger Maestro, v12.8). The CRISPR/Cas9-induced genetic KO of CYP1A1 and CYP3A28 was achieved in BFH12 cells using RNP-complex approach. The gene deletion was confirmed by Sanger sequencing, qPCR and/or immunoblotting. Therefore, the cytotoxic effects of AFB1 on native and CYP1A1/CYP3A28 KO BFH12 cells was evaluated using the WST-1 reagent. Results: Docking of AFB1 onto CYP1A1 resulted in two relevant binding modes. In the first one, a hydrogen bond (i.e., ASN226-ring A) and π- π stacking interactions (i.e., PHE228-ring B and PHE127-ring C) were showed, suggesting the formation of the endo-epoxide metabolite. The second pose (π- π stacking interactions between PHE127-ring B) suggests the production of exo-epoxide derivative. Docking of AFB1 onto CYP3A28 resulted in SER119-ring A hydrogen bond, possibly allowing the formation of AFQ1 derivative. CYP1A1 and CYP3A28 CRISPR/Cas9-mediated deletion was confirmed by Sanger sequencing. In engineered cells, CYP1A1 apoprotein was reduced of ~80% and CYP3A28 mRNA expression was completely ablated. Compared to native cells, AFB1 cytotoxicity was significantly reduced in CYP1A1 KO cells, while it was unmodified in CYP3A28 KO cells, corroborating docking predictions and suggesting the main involvement of CYP1A1 in AFB1 bioactivation. Conclusions: This study, through the integration of molecular docking and genetic KO approaches, laid the groundwork to decipher the role played by CYP1A1 and CYP3A28 in AFB1 metabolism and bioactivation. Further studies (i.e., AFB1 metabolite profiling and RNA-seq analysis) are envisaged to implement the knowledge on AFB1 fate in bovine liver