Regulation of the Methylation and Expression Levels of the BMPR2 Gene by SIN3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension.

BACKGROUND: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. METHODS: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. RESULTS: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. CONCLUSIONS: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.

Non-linear optical imaging of atherosclerotic plaques in the context of SIV and HIV infection prominently detects crystalline cholesterol esters.

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation.

Impact of coxsackievirus-B4E2 combined with a single low dose of streptozotocin on pancreas of outbred mice: investigation of viral load, pathology and inflammation.

Coxsackieviruses B (CV-B) belong to the EV-B species. CV-B and particularly CV-B4 are thought to be involved in the development of chronic diseases like type 1 diabetes (T1D). The mechanisms of the enteroviral pathogenesis of T1D are not well known, yet. The in vitro studies are rich with information but in vivo infection models are needed to investigate the impact of viruses onto organs. Our objective was to study the impact of CV-B4E2 combined with a single sub-diabetogenic dose of streptozotocin (STZ) on the pancreas of mice. The infection with CV-B4E2 of CD1 outbred mice treated with a sub-diabetogenic dose of STZ induced hyperglycemia and hypoinsulinemia. Along with the chemokine IP-10, viral RNA and infectious particles were detected in the pancreas. The pancreas of these animals was also marked with insulitis and other histological alterations. The model combining STZ and CV-B4E2 opens the door to new perspectives to better understand the interactions between virus and host, and the role of environmental factors capable, like STZ, to predispose the host to the diabetogenic effects of enteroviruses.

Inhibition of coxsackievirus B4 by Lactobacillus plantarum.

The enterovirus Coxsackievirus B4 (CV-B4) can infect different human tissues and provoke abnormal function or destruction of various organs and cells. Moreover, its infections have been linked to the onset of type 1 diabetes. Coxsackievirus B4 is classified as a "challenging virus", due to the intense yet vain efforts to find effective prevention and therapeutic agents, especially within biological compounds. Lactobacillus plantarum is a lactic acid bacterium that is endowed with probiotic properties, and holds great potential for applications in medical and food industry sectors. Several compounds produced by this microorganism have been associated with various benefits including antimicrobial activity. In this work, we investigated the possible antiviral abilities of two Lb. plantarum strains and their derivatives against CV-B4. The different assays carried out (e.g. pre-incubation, competition and post-infection, using HEp-2 cells as human cell model) suggest that the tested microorganisms and their derivatives have an in vitro inhibiting activity against CV-B4. This is the first report showing the anti-CVB4 activity of Lb. plantarum strains and their derivatives.

A Calsequestrin Cis-Regulatory Motif Coupled to a Cardiac Troponin T Promoter Improves Cardiac Adeno-Associated Virus Serotype 9 Transduction Specificity.

Adeno-associated virus serotype 9 (AAV9) is an efficient vector for gene transfer to the myocardium. However, the use of ubiquitous promoters, such as the cytomegalovirus (CMV) promoter, can result in expression of the transgene in organs other than the heart. This study tested if the efficiency and specificity of cardiac transcription from a chicken cardiac troponin T (TnT) promoter could be further increased by incorporating a cardiomyocyte-specific transcriptional cis-regulatory motif from human calsequestrin 2 (CS-CRM4) into the expression cassette (Enh.TnT). The efficiency of luciferase expression from the TnT and Enh.TnT constructs was compared to expression of luciferase under the control of the CMV promoter in both adult and neonatal mice. Overall, expression levels of luciferase in the heart were similar in mice injected with AAV9.TnT.Luc, AAV9.Enh.TnT.Luc and AAV9.CMV.Luc. In contrast, expression levels of luciferase activity in nontarget organs, including the liver and muscle, was lower in mice injected with the AAV9.TnT.Luc compared to AAV9.CMV.Luc and was negligible with AAV9.Enh.TnT. In neonates, in organs other than the heart, luciferase expression levels were too low to be quantified for all constructs. Taken together, the data show that the AAV9 Enh.TnT constructs drives high levels of expression of the transgene in the myocardium, with insignificant expression in other organs. These properties reduce the risks associated with the AAV9-mediated expression of the therapeutic protein of interest in nontarget organs. The excellent cardiac specificity should allow for the use of higher vector doses than are currently used, which might be essential to achieve the levels of transgene expression necessary for therapeutic benefits. Taken together, the findings suggest that the Enh.TnT transcription unit is a potentially attractive tool for clinical cardiac gene therapy in adults.

Protein S Protects against Podocyte Injury in Diabetic Nephropathy.

Background Diabetic nephropathy (DN) is a leading cause of ESRD in the United States, but the molecular mechanisms mediating the early stages of DN are unclear.Methods To assess global changes that occur in early diabetic kidneys and to identify proteins potentially involved in pathogenic pathways in DN progression, we performed proteomic analysis of diabetic and nondiabetic rat glomeruli. Protein S (PS) among the highly upregulated proteins in the diabetic glomeruli. PS exerts multiple biologic effects through the Tyro3, Axl, and Mer (TAM) receptors. Because increased activation of Axl by the PS homolog Gas6 has been implicated in DN progression, we further examined the role of PS in DN.Results In human kidneys, glomerular PS expression was elevated in early DN but suppressed in advanced DN. However, plasma PS concentrations did not differ between patients with DN and healthy controls. A prominent increase of PS expression also colocalized with the expression of podocyte markers in early diabetic kidneys. In cultured podocytes, high-glucose treatment elevated PS expression, and PS knockdown further enhanced the high-glucose-induced apoptosis. Conversely, PS overexpression in cultured podocytes dampened the high-glucose- and TNF-α-induced expression of proinflammatory mediators. Tyro3 receptor was upregulated in response to high glucose and mediated the anti-inflammatory response of PS. Podocyte-specific PS loss resulted in accelerated DN in streptozotocin-induced diabetic mice, whereas the transient induction of PS expression in glomerular cells in vivo attenuated albuminuria and podocyte loss in diabetic OVE26 mice.Conclusions Our results support a protective role of PS against glomerular injury in DN progression.

Coxsackievirus B4 vertical transmission in a murine model.

BACKGROUND: Life-threatening infections with type B Coxsackieviruses (CV-B) are frequently encountered among newborns and are partly attributed to vertically-transmitted virus. Our current study investigates this alternative way of contamination by CV-B, using a mouse model. METHODS: Pregnant Swiss mice were intraperitoneally inoculated with CV-B4 E2 at gestational day 10(G) or 17G. Dams and offspring were monitored for mortality and morbidity, and sampled at different time-points to document the infection and explore eventual vertical transmission. RESULTS: Inoculation at day 10G induced an important rate of abortion and a decrease in the number of delivered pups per litter, whereas inoculation at day 17G was marked by preterm delivery and significant behavioral changes in dams. Only one case of spastic paralysis and one case of pancreatitis were recorded among surviving pups. Seroneutralization revealed anti-CV-B4 neutralizing antibodies in infected dams and their partial transfer to offspring. Viral genome detection by RT-PCR and viral progeny titration in several tissues (dams' uteri, amniotic sac, amniotic fluid, placenta, umbilical cord, pancreas and heart) attested and documented CV-B4 vertical transmission to the majority of analyzed offspring. Virus detection in fetuses suggests transplacental transmission, but perinatal transmission during delivery could be also suggested. Vertically transmitted CV-B might even persist since prolonged viral RNA detection was noticed in the pancreas and heart from offspring born to dams inoculated at day 17G. CONCLUSION: This model of CV-B4 vertical transmission in mice, in addition to allow a better understanding of CV-B infections in fetuses and newborns, constitutes a useful tool to investigate the pathogenesis of CV-B associated chronic diseases.

Anti-coxsackievirus B4 (CV-B4) enhancing activity of serum associated with increased viral load and pathology in mice reinfected with CV-B4.

In previous studies it was shown that inoculation of Swiss albino mice with CV-B4 E2 resulted in the production of serum IgG capable of enhancing the CV-B4 E2 infection of murine spleen cells cultures. To investigate whether such an enhancing activity of serum can play a role in vivo, we decided to study the CV-B4 E2 infection in mice exposed to successive inoculations of virus. In Swiss albino mice infected with CV-B4 E2 at the age of 21 days, anti-CV-B4 E2 neutralizing and enhancing activities of their serum peaked after 55 d. In contrast, mice inoculated at the age of 55 d expressed much lower activities. Despite the neutralizing activity of serum, CV-B4 E2 inoculated a second time to 55 day-old animals spread into the host. At the age of 72 and 89 d the levels of viral RNA and infectious particles were higher in organs of animals exposed to 2 successive infections compared with animals infected once at the age of 21 d or 55 d. In animals with 2 successive inoculations of CV-B4 E2 there was a relationship between the anti-CV-B4 E2 enhancing activity of serum and the level of viral RNA in organs and an enhancement of pathology was observed as displayed by histological analysis of pancreas and hyperglycaemia. Altogether our data strongly suggest that an anti-CV-B4 E2 enhancing activity in the host can play a role in the outcome of a secondary infection with this virus.

Central nervous system infection following vertical transmission of Coxsackievirus B4 in mice.

Coxsackie B viruses (CV-B) are important pathogens associated with several central nervous system (CNS) disorders. CV-B are mainly transmitted by the faecal-oral route, but there is also evidence for vertical transmission. The outcome of in utero CV-B infections on offspring's CNS is poorly explored. The aim of this study was to investigate vertical transmission of CV-B to the CNS. For this purpose, pregnant Swiss albino mice were intraperitoneally inoculated with CV-B4 E2 at gestational days 10G or 17G. Different CNS compartments were collected and analyzed for virus infection and histopathological changes. Using plaque assays, we demonstrated CV-B4 E2 vertical transmission to offspring's CNS. Viral RNA persisted in the CNS up to 60 days after birth, as evidenced by a sensitive semi-nested(sn) reverse transcripton(RT)-PCR method. This was despite infectious particles becoming undetectable at later time points. Persistence was associated with inflammatory lesions, lymphocyte infiltration and viral dsRNA detected by immunohistochemistry. Offspring born to dams mock- or virus-infected at day 17G were challenged by the same virus at day 21 after birth (-+ and ++ groups, respectively). Sn-RT-PCR and histology results compared between both ++ and -+ groups, show that in utero infection did not enhance CNS infection during challenge of the offspring with the same virus.

Immunoglobulin G-dependent enhancement of the infection with Coxsackievirus B4 in a murine system.

It was demonstrated that specific IgG can enhance the infection with CV-B4, in vitro, in the human system. This enhancement could be involved in the pathophysiology of CV-B4 induced diseases. To investigate further the role of enhancing IgG in the infection with CV-B4 E2 in vivo, animal models are needed. Therefore, it was decided to assess whether inoculation of CV-B4 E2 to mice results in the appearance of IgG able to enhance the infection with this virus. Swiss albino mice were inoculated with CV-B4 E2 intraperitoneally. Serum samples were obtained from tail vein blood collected from day 0 to day 80 p.i. IgG were isolated by Protein G affinity chromatography. Seroneutralisation assays were carried out. In total murine spleen cells cultures inoculated with CV-B4 E2 mixed with various dilutions of serum or IgG samples, the enhancing activity was assayed through i) the antiviral activity titer of supernatants ii) the detection of intracellular viral RNA by RT-PCR iii) the level of infectious particles in supernatants. In most serum samples (76/105), neutralizing and enhancing activities were detected peaking between days 14 and 30 p.i and were higher in sera from mice inoculated with 2.10(6) TCID50 units than with lower doses. The enhancing activity was due to the IgG-enriched fraction of serum from CV-B4 E2 infected animals but not from control animals. These data show that IgG from immune mice can enhance the infection of splenocytes with CV-B4 E2 in vitro and open the way to explore whether such an enhancing activity can play a role in vivo.