Sex-Specific Effects of Plastic Caging in Murine Viral Myocarditis.

BACKGROUND: Myocarditis is an inflammatory heart disease caused by viral infections that can lead to heart failure, and occurs more often in men than women. Since animal studies have shown that myocarditis is influenced by sex hormones, we hypothesized that endocrine disruptors, which interfere with natural hormones, may play a role in the progression of the disease. The human population is exposed to the endocrine disruptor bisphenol A (BPA) from plastics, such as water bottles and plastic food containers. METHODS: Male and female adult BALB/c mice were housed in plastic versus glass caging, or exposed to BPA in drinking water versus control water. Myocarditis was induced with coxsackievirus B3 on day 0, and the endpoints were assessed on day 10 post infection. RESULTS: We found that male BALB/c mice that were exposed to plastic caging had increased myocarditis due to complement activation and elevated numbers of macrophages and neutrophils, whereas females had elevated mast cell activation and fibrosis. CONCLUSIONS: These findings show that housing mice in traditional plastic caging increases viral myocarditis in males and females, but using sex-specific immune mechanisms.

BPA Alters Estrogen Receptor Expression in the Heart After Viral Infection Activating Cardiac Mast Cells and T Cells Leading to Perimyocarditis and Fibrosis.

Myocarditis is an inflammatory heart disease that leads to dilated cardiomyopathy (DCM) and heart failure. Sex hormones play an important role in the development of myocarditis with testosterone driving disease in males and estrogen being cardioprotective in females. The human population is widely exposed to the endocrine disruptor bisphenol A (BPA) from plastics such as water bottles, plastic food containers, copy paper, and receipts. Several clinical and numerous animal studies have found an association between elevated BPA levels and cardiovascular disease. A recent report found elevated levels of BPA in the serum of patients with DCM compared to healthy controls. In this study we examined whether exposure to BPA for 2 weeks prior to viral infection and leading up to myocarditis at day 10 altered inflammation in female BALB/c mice housed in standard plastic cages/water bottles with soy-free food and bedding. We found that a human relevant dose of BPA (25 µg/L) in drinking water, with an estimated exposure of 5 µg BPA/kg BW, significantly increased myocarditis and pericarditis compared to control water without altering viral genome levels in the heart. BPA exposure activated ERα and ERß in the spleen 24 h after infection and phosphorylated ERα and ERß during myocarditis, but decreased ERα and increased ERß mRNA in the heart as measured by qRT-PCR. Exposure to BPA significantly increased CD4+ T cells, IFNγ, IL-17A, TLR4, caspase-1, and IL-1ß in the heart. BPA exposure also increased cardiac fibrosis compared to controls. Mast cells, which are associated with cardiac remodeling, were found to increase in number and degranulation, particularly along the pericardium. Interestingly, plastic caging/water bottle exposure alone led to increased mast cell numbers, pericardial degranulation and fibrosis in female BALB/c mice compared to animals housed in glass cages/water bottles with soy-free food and bedding. These data suggest that BPA exposure may increase the risk of developing myocarditis after a viral infection in women.

In vitro oxygen exposure promotes maturation of the oxygen sensitive contraction in pre-term chicken ductus arteriosus.

The ductus arteriosus (DA) are O2-sensitive, embryonic blood vessels that serve as a right-to-left shunt in developing avian embryos. Prior to internal pipping, the chicken DA produces a weak O2-induced contraction. During hatching, the O2-sensitivity of the avian DA vessels increases significantly. To see if we could accelerate the maturation of chicken DA O2-sensitivity, we exposed the vessel in vitro to elevated O2 (25 kPa) for 3-h prior to internal pipping on day 19 of incubation. The DA initially responded to increasing O2 with a weak contraction (0.15±0.04 N/m) that significantly increased in strength (0.63±0.06 N/m) during 3-h 25 kPa O2 exposure. A tonic influence of nitric oxide, not present at low O2, appeared during the 3-h 25 kPa O2 exposure. The long-term O2-induced contraction was mediated by both L-type Ca(2+) channels and internal Ca(2+) stores. The Rho-kinase pathway inhibitors Y-27632 and fasudil produced significant relaxation, suggesting a role for Ca(2+) sensitization in the contractile response to the 3h of elevated O2. While the day 19 DA initially exhibited an immature contractile response to O2, maturation of the pathways regulating O2-induced contraction was accelerated by exposure to 25 kPa O2, producing contractions similar in magnitude to those found during the final stage of hatching. This suggests that maturation of O2-sensitivity may be accelerated in vivo by increasing arterial O2 levels.

Inducible macropinocytosis of hyaluronan in B16-F10 melanoma cells.

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Endocytosis is thought to play an essential role in the catabolism of HA due to the intracellular compartmentalization of the HA degrading hyaluronidase enzymes. Previous investigations have shown that keratinocytes, chondrocytes and breast tumor cell lines endocytose HA via the cell surface glycoprotein, CD44. However, other cell types endocytose HA using a CD44-independent mechanism that remains to be defined. The purpose of this study was to investigate HA endocytosis in B16-F10 melanoma cells. We found that B16-F10 melanoma cells expressed CD44 on their surfaces. Unexpectedly, CD44 did not play a role in the endocytosis of HA. Electron microscopy studies revealed that B16-F10 melanoma cells exhibited membrane ruffling, a characteristic feature of macropinocytosis, only after incubating the cells with the HA co-polymer. Moreover, B16-F10 melanoma cells endocytosed HA via macropinocytosis as assessed by drug inhibition studies and the co-localization of fluorescently labeled HA with fluorescent tracers under confocal microscopy. Based on these results, we conclude that induced macropinocytosis may provide a previously unrecognized avenue for HA endocytosis in some cell types.

Mechanisms mediating the oxygen-induced vasoreactivity of the ductus arteriosus in the chicken embryo.

The avian embryo provides a novel model for studying the ductus arteriosus (DA) during the transition from in ovo to ex ovo life. Here we examined the mechanisms regulating the vasoreactivity of the two morphologically distinct portions of the chicken DA (proximal and distal) in response to O(2). Oxygen-induced contraction is redox sensitive and reversed by the reducing agent dithiothreitol and the H(2)O(2) scavenger N-mercaptopropionylglycine. As in the mammalian DA, inhibiting mitochondrion-derived reactive oxygen species production with rotenone and antimycin A relaxed the O(2)-constricted DA. The contractile response to O(2) matures during hatching and is mimicked by the K(v) channel inhibitor 4-aminopyridine (4-AP) on day 19 and externally pipped (EP) embryos. Together, O(2) and 4-AP significantly increase DA tone above that observed with either alone. The O(2)-induced contraction is mediated by influx of extracellular Ca(2+) through l-type Ca(2+) and store-operated channels. Inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores play a minor role in the O(2)-induced contraction. The O(2)-induced contraction is mediated by the Rho kinase pathway, as fasudil and Y-27632 significantly relax the O(2) contracted DA. Prostaglandins E(2), F(2alpha), and D(2) produce significant contraction of the proximal DA. The O(2)-induced relaxation of the distal portion of the DA is mediated by an endothelial-derived nitric oxide/cGMP pathway. Both 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and endothelial cell removal inhibit O(2)-induced relaxation in the distal segment. Mechanisms regulating O(2)-induced contraction in chicken proximal DA are similar to those found in mammalian DA, making the chicken a useful model for studying development of this O(2)-sensitive vessel.

Maturation of the contractile response of the Emu ductus arteriosus.

The avian embryo has a pair of ductus arteriosi that allow the blood to bypass the pulmonary circulation prior to the initiation of lung ventilation. Our objective was to characterize the factors regulating DA tone during the later stages of development in the emu embryo. We examined in vitro the reactivity of the emu ductus from day 39 through 49 of a 50-day incubation. Steady state tension was not altered by the COX inhibitor indomethacin or the nitric oxide synthase inhibitor L-NAME. However, prostaglandin E(2) (PGE(2)) produced a significant relaxation. Norephinephrine and U-46619 produced strong significant contractions in the emu DA and the adrenergic response matured with development. The contractile response to oxygen matured as the embryo developed with significant oxygen-induced contraction on days 45 and 49, but not on day 39 of incubation. The Kv channel inhibitor 4-aminopyridine induced the contraction of the day 48-49 ductus of similar magnitude as the oxygen-induced contraction. The oxygen-induced contraction was reversed by the reducing agent DTT and the electron transport chain inhibitor rotenone. These results suggest that while the emu DA responds to PGE(2), locally produced PGE(2) are not the important regulators of vessel tone. Additionally, relaxation upon addition of the mitochondria electron transport chain inhibitor rotenone suggests that the mitochondria might be acting as vascular oxygen sensors in this system through the production of reactive oxygen species to stimulate the oxygen-induced contraction in a similar fashion to mammals.