An elevated plasma concentration of serotonin ([5-HT]) is a common feature of cardiovascular disease often associated with enhanced platelet activation and thrombosis. Whether elevated in vivo plasma 5-HT per se represents an independent risk factor for platelet hyperreactivity or only is an epiphenomenon of cardiovascular disease is poorly understood. We examined in vitro and in vivo platelet function following a 24 hr elevation of plasma [5-HT] in mice. In vivo administration of 5-HT using osmotic minipumps increased plasma [5-HT] in treated mice compared to control mice instrumented with saline loaded pumps. 5-HT infusion did not increase systolic blood pressure, but markers of platelet activation including P-selectin and PEJon/A staining were increased and these findings coincided with the enhanced aggregation of isolated platelets in response to type I fibrillar collagen. Tail bleeding times and the time to occlusion following chemical damage to the carotid artery were shortened in 5-HT-infused mice. 5-HT-infused mice were treated with paroxetine (Prx) to block 5-HT uptake via the serotonin transporter (SERT). Prx lowered platelet [5-HT] and attenuated platelet activation and aggregation. These results and our biochemical indices of enhanced 5-HT intracellular signaling in the platelets of 5-HT-infused mice reveal a mechanistic link between elevated plasma [5-HT], abnormal intracellular 5-HT signaling and accentuated platelet aggregation. Although a down-regulation of the serotonin transporter (SERT) on the platelet surface may counteract the pro-thrombotic influence of elevated plasma [5HT], this compensatory mechanism may fail to prevent the increased thrombotic risk caused by elevated plasma [5-HT].

Cardiotoxicity is one of the major side effects encountered during cancer chemotherapy with doxorubicin (DOX) and other anthracyclines. Previous studies have shown that oxidative stress caused by DOX is one of the primary mechanisms for its toxic effects on the heart. Since the redox-sensitive transcription factor, Nrf2, plays a major role in protecting cells from the toxic metabolites generated during oxidative stress, we examined the effects of the phytochemical sulforaphane (SFN), a potent Nrf2-activating agent, on DOX-induced cardiotoxicity. These studies were carried out both in vitro and in vivo using rat H9c2 cardiomyoblast cells and wild type 129/sv mice, and involved SFN pretreatment followed by SFN administration during DOX exposure. SFN treatment protected H9c2 cells from DOX cytotoxicity and also resulted in restored cardiac function and a significant reduction in DOX-induced cardiomyopathy and mortality in mice. Specificity of SFN induction of Nrf2 and protection of H9c2 cells was demonstrated in Nrf2 knockdown experiments. Cardiac accumulation of 4-hydroxynonenal (4-HNE) protein adducts, due to lipid peroxidation following DOX-induced oxidative stress, was significantly attenuated by SFN treatment. The respiratory function of cardiac mitochondria isolated from mice exposed to DOX alone was repressed, while SFN treatment with DOX significantly elevated mitochondrial respiratory complex activities. Co-administration of SFN reversed the DOX-associated reduction in nuclear Nrf2 binding activity and restored cardiac expression of Nrf2-regulated genes, at both the RNA and protein levels. Together, our results demonstrate for the first time that the Nrf2 inducer, SFN, has the potential to provide protection against DOX-mediated cardiotoxicity.

Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation.

Serotonin (5-HT) transporter (SERT) regulates the level of 5-HT in placenta. Initially, we found that in gestational diabetes mellitus (GDM), whereas free plasma 5-HT levels were elevated, the 5-HT uptake rates of trophoblast were significantly down-regulated, due to impairment in the translocation of SERT molecules to the cell surface. We sought to determine the factors mediating the down-regulation of SERT in GDM trophoblast. We previously reported that an endoplasmic reticulum chaperone, ERp44, binds to Cys200 and Cys209 residues of SERT to build a disulfide bond. Following this posttranslational modification, before trafficking to the plasma membrane, SERT must be dissociated from ERp44; and this process is facilitated by insulin signaling and reversed by the insulin receptor blocker AGL2263. However, the GDM-associated defect in insulin signaling hampers the dissociation of ERp44 from SERT. Furthermore, whereas ERp44 constitutively occupies Cys200/ Cys209 residues, one of the SERT glycosylation sites, Asp208 located between the two Cys residues, cannot undergo proper glycosylation, which plays an important role in the uptake efficiency of SERT. Herein, we show that the decrease in 5-HT uptake rates of GDM trophoblast is the consequence of defective insulin signaling, which entraps SERT with ERp44 and impairs its glycosylation. In this regard, restoring the normal expression of SERT on the trophoblast surface may represent a novel approach to alleviating some GDM-associated complications.serotonin | ERp44 | insulin | serotonin transporter | gestational diabetes mellitus

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Purpose: Astronauts traveling beyond low-Earth orbit will be exposed to high linear-energy transfer charged particles. Because there is concern about the adverse effects of space radiation on the cardiovascular system, this study assessed cardiac function and structure and immune cell infiltration in a mouse model of charged-particle irradiation.Materials and methods: Male C57BL/6J mice were exposed to oxygen ions ( 16 O, 600 MeV/n at 0.25−0.26 Gy/min to a total dose of 0, 0.05, 0.1, 0.25, or 1 Gy), protons (150 MeV, 0.35−0.55 Gy/min to 0, 0.5, or 1 Gy), or protons (150 MeV, 0.5 Gy) followed by 16 O (600 MeV/n, 0.1 Gy). Separate groups of mice received 137 Cs γ-rays (1 Gy/min to 0, 0.5, 1, or 3 Gy) as a reference. Cardiac function and blood velocity were measured with ultrasonography at 3, 5, 7, and 9 months after irradiation. At 2 weeks, 3 months, and 9 months, cardiac tissue was collected to assess apoptosis, tissue remodeling, and markers of immune cells.