Bisphenol A (BPA) is found in human urine and fat tissue. Higher urinary BPA concentrations are associated with arterial hypertension. To shed light on the underlying mechanism, we orally administered BPA (4 nM to 400 μM in drinking water) to 8-wk-old CD11 mice over 30 d. Mice developed dosage-dependent high blood pressure (systolic 130 ± 12 vs. 170 ± 12 mmHg; EC50 0.4 μM), impairment of acetylcholine (AcH)-induced carotid relaxation (0.66 ± 0.08 vs. 0.44 ± 0.1 mm), a 1.7-fold increase in arterial angiotensin II (AngII), an 8.7-fold increase in eNOS mRNA and protein, and significant eNOS-dependent superoxide and peroxynitrite accumulation. AngII inhibition with 0.5 mg/ml losartan reduced oxidative stress and normalized blood pressure and endothelium-dependent relaxation, which suggests that AngII uncouples eNOS and contributes to the BPA-induced endothelial dysfunction by promoting oxidative and nitrosative stress. Microarray analysis of mouse aortic endothelial cells revealed a 2.5-fold increase in expression of calcium/calmodulin-dependent protein kinase II-α (CaMKII-α) in response to 10 nM BPA, with increased expression of phosphorylated-CaMKII-α in carotid rings of BPA-exposed mice, whereas CaMKII-α inhibition with 100 nM autocamptide-2-related inhibitor peptide (AIP) reduced BPA-mediated increase of superoxide. Administration of CaMKII-α inhibitor KN 93 reduced BPA-induced blood pressure and carotid blood velocity in mice, and reverted BPA-mediated carotid constriction in response to treatment with AcH. Given that CaMKII-α inhibition prevents BPA-mediated high blood pressure, our data suggest that BPA regulates blood pressure by inducing AngII/CaMKII-α uncoupling of eNOS.

Inhibition of extracellular matrix (ECM) degradation may represent a mechanism for cardiac protection against ischemia. Extracellular matrix metalloproteinase inducer (EMMPRIN) is highly expressed in response to acute myocardial infarction (AMI), and induces activation of several matrix metalloproteinases (MMPs), including gelatinases MMP-2 and MMP-9. We targeted EMMPRIN with paramagnetic/fluorescent micellar nanoparticles conjugated with the EMMPRIN binding peptide AP-9 (NAP9), or an AP-9 scrambled peptide as a negative control (NAPSC). We found that NAP9 binds to endogenous EMMPRIN in cultured HL1 myocytes and in mouse hearts subjected to ischemia/reperfusion (IR). Injection of NAP9 at the time of or one day after IR, was enough to reduce progression of myocardial cell death when compared to Control and NAPSC injected mice (infarct size in NAP9 injected mice: 32%±6.59 vs Control: 46%±9.04 or NAPSC injected mice: 48%±7.64). In the same way, cardiac parameters were recovered to almost healthy levels (LVEF NAP9 63% ± 7.24 vs Control 42% ± 4.74 or NAPSC 39% ± 6.44), whereas ECM degradation was also reduced as shown by inhibition of MMP-2 and MMP-9 activation. Cardiac magnetic resonance (CMR) scans have shown a signal enhancement in the left ventricle of NAP9 injected mice with respect to non-injected, and to mice injected with NAPSC. A positive correlation between CMR enhancement and Evans-Blue/TTC staining of infarct size was calculated (R:0.65). Taken together, these results point to EMMPRIN targeted nanoparticles as a new approach to the mitigation of ischemic/reperfusion injury.

Epidemiological studies link long term exposure to xenoestrogen Bisphenol-A to adverse cardiovascular effects. Our previous results show that BPA induces hypertension by a mechanism involving CamKII activation and increased redox stress caused by eNOS uncoupling. Recently, CamKII sustained activation has been recognized as a central mediator of programmed cell death in cardiovascular diseases, including necroptosis. However, the role of necroptosis in cardiac response to BPA had not yet been explored. Mice exposed to BPA for 16 weeks showed altered heart function, electrical conduction, and increased blood pressure. Besides, a stress test showed ST-segment depression, indicative of cardiac ischemia. The hearts exhibited cardiac hypertrophy and reduced vascularization, interstitial edema, and large hemorrhagic foci accompanied by fibrinogen deposits. BPA initiated a cardiac inflammatory response, up-regulation of M1 macrophage polarization, and increased oxidative stress, coinciding with the increased expression of CamKII and the necroptotic effector RIP3. In addition, cell death was especially evident in coronary endothelial cells within hemorrhagic areas, and Evans blue extravasation indicated a vascular leak in response to Bisphenol-A. Consistent with the in vivo findings, BPA increased the necroptosis/apoptosis ratio, the expression of RIP3, and CamKII activation in endothelial cells. Necrostatin-1, an inhibitor of necroptosis, alleviated BPA induced cardiac dysfunction and prevented the inflammatory and hemorrhagic response in mice. Mechanistically, silencing of RIP3 reversed BPA-induced necroptosis and CamKII activation in endothelial cells, while inhibition of CamKII activation by KN-93 had no effect on RIP3 expression but decreased necroptotic cell death suggesting that BPA induced necroptosis is mediated by a RIP 3/CamKII dependent pathway. Our results reveal a novel pathogenic role of BPA on the coronary circulation. BPA induces endothelial cell necroptosis, promotes the weakening of coronary vascular wall, which caused internal ventricular hemorrhages, delaying the reparative process and ultimately leading to cardiac dysfunction.

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