The interplay between sex, time of day, fasting status, and their impact on cardiac mitochondrial structure, function, and dynamics.

Mitochondria morphology and function, and their quality control by mitophagy, are essential for heart function. We investigated whether these are influenced by time of the day (TOD), sex, and fed or fasting status, using transmission electron microscopy (EM), mitochondrial electron transport chain (ETC) activity, and mito-QC reporter mice. We observed peak mitochondrial number at ZT8 in the fed state, which was dependent on the intrinsic cardiac circadian clock, as hearts from cardiomyocyte-specific BMAL1 knockout (CBK) mice exhibit different TOD responses. In contrast to mitochondrial number, mitochondrial ETC activities do not fluctuate across TOD, but decrease immediately and significantly in response to fasting. Concurrent with the loss of ETC activities, ETC proteins were decreased with fasting, simultaneous with significant increases of mitophagy, mitochondrial antioxidant protein SOD2, and the fission protein DRP1. Fasting-induced mitophagy was lost in CBK mice, indicating a direct role of BMAL1 in regulating mitophagy. This is the first of its kind report to demonstrate the interactions between sex, fasting, and TOD on cardiac mitochondrial structure, function and mitophagy. These studies provide a foundation for future investigations of mitochondrial functional perturbation in aging and heart diseases.

Extracellular vesicles produced by human-induced pluripotent stem cell-derived endothelial cells can prevent arterial stenosis in mice via autophagy regulation.

Intravascular transplantation of human-induced pluripotent stem cells (hiPSCs) demonstrated a significant therapeutic effect in the treatment of restenosis by the paracrine function of extracellular vesicles (EVs). However, the risk of tumorigenicity and poor cell survival limits its clinical applications. In this study, we for the first time applied a highly efficient and robust three-dimensional (3D) protocol for hiPSC differentiation into endothelial cells (ECs) with subsequent isolation of EVs from the derived hiPSC-EC (ECs differentiated from hiPSCs), and validated their therapeutic effect in intimal hyperplasia (IH) models. We found that intravenously (iv) injected EVs could accumulate on the carotid artery endothelium and significantly alleviate the intimal thickening induced by the carotid artery ligation. To elucidate the mechanism of this endothelial protection, we performed miRNA expression profiling and found out that among the most conserved endothelial miRNAs, miR-126 was the most abundant in hiPSC-EC-produced EVs (hiPSC-EC-EV). MiR-126 depletion from hiPSC-EC-EV can hinder its protective effect on human umbilical vein endothelial cells (HUVECs) in an inflammatory process. A variety of functional in vitro studies revealed that miR-126 was able to prevent endothelial apoptosis after inflammatory stimulation, as well as promote EC migration and tube formation through autophagy upregulation. The latter was supported by in vivo studies demonstrating that treatment with hiPSC-EC-EV can upregulate autophagy in mouse carotid artery ECs, thereby preventing IH and modulating vascular homeostasis via remodeling of the vascular intima. Our findings suggest a regulatory mechanism for the therapeutic effect on arterial restenosis by autophagy regulation, and provide a potential strategy for clinical treatment of the disease.

Geospatial characterization of rural settlements and potential targets for revitalization by geoinformation technology.

To better implement the Strategy of Rural Revitalization, it is essential to characterize the rural settlements and understand their roles in the socio-environmental interactive system. This paper is hence aimed at achieving such a study using different spatial analysis such as kernel density and spatial autocorrelation (SA) and modeling approaches, e.g., simple and multiple linear regression analyses taking Jiangxi, a province in China as an example. Remote sensing, topographic and socioeconomic data were employed for this purpose. Through these analyses, it is found that the rural settlements in the study area appear to have a spatial distribution pattern of "dense north and sparse south" as an "F" type, and are quantitatively characterized as low elevations, flat terrain, high river and road densities, rich cultivated land resources and susceptible to the impact of urban radiation with a R2 of 0.520-0.748. Based on this understanding, a new inequality evaluation indicator of rural development, i.e., socio-environmental evaluation index (SEI), was developed. Areas with SEI lower than 0.40 should be given a priority to implement the revitalization strategy in the province. This index can also be extended to study of the imbalance of rural development in other regions and countries.

Sustained Release of MiR-217 Inhibitor by Nanoparticles Facilitates MSC-Mediated Attenuation of Neointimal Hyperplasia After Vascular Injury.

Mesenchymal stem cells (MSCs) have been proven capable of differentiating into endothelial cells (ECs) and increasing vascular density in mouse ischemia models. However, the therapeutic potential of MSCs in neointimal hyperplasia after vascular injury is still not fully understood. In this study, we proposed that sustained release of miR-217 inhibitor encapsulated by nanoparticles in MSCs can enhance the therapeutic effects of MSCs on alleviating neointimal hyperplasia in a standard mouse wire injury model. We intravenously administered MSCs to mice with injured arteries and examined neointimal proliferation, endothelial differentiation and senescence. We demonstrated that MSCs localized to the luminal surface of the injured artery within 24 h after injection and subsequently differentiated into endothelial cells, inhibited neointimal proliferation and migration of vascular smooth muscle cells. Transfection of MSCs with poly lactic-co-glycolic acid nanoparticles (PLGA-NP) encapsulating an miR-217 agomir abolished endothelial differentiation as well as the therapeutic effect of MSCs. On the contrary, silencing of endogenous miR-217 improved the therapeutic efficacy of MSCs. Our study provides a new strategy of augmenting the therapeutic potency of MSCs in treatment of vascular injury.

HO­1 knockdown upregulates the expression of VCAM­1 to induce neutrophil recruitment during renal ischemia­reperfusion injury.

Heme oxygenase­1 (HO­1) has been reported to be upregulated following renal ischemia­reperfusion injury (IRI) and plays a key cytoprotective role; however, the underlying molecular mechanisms of its protective effects remain poorly understood. In the present study, in order to further elucidate the molecular mechanisms underlying the cytoprotective role of HO­1 in renal IRI, HO­1+/+ and HO­1+/­ mice were subjected to renal ischemia and subsequent reperfusion followed by the analysis of blood urea nitrogen (BUN) and serum creatinine (SCr) levels, the severity of histological changes, HO­1 and vascular cell adhesion molecule­1 (VCAM­1) protein expression, the mRNA expression of inflammatory factors and the effects of VCAM­1 blockade. The results of the present study demonstrated that the upregulated expression levels of VCAM­1 in HO­1+/­ mice during IRI increased the extent of renal tissue damage and activated the inflammatory response. These effects were subsequently reversed following infusion with an anti­VCAM­1 antibody. In addition, the upregulated expression of VCAM­1 in mouse glomerulus vascular endothelial cells isolated from HO­1+/­ mice increased the adhesion and migration of neutrophils, effects which were also reversed upon incubation with an anti­VCAM­1 antibody. These results indicated that HO­1 knockdown may upregulate the expression of VCAM­1 during renal IRI, resulting in increased neutrophil recruitment and the activation of the inflammatory response, thereby exacerbating renal IRI. The present study thus highlights the regulatory mechanisms of HO­1 in renal IRI and provides a potential target for the clinical treatment of IRI following renal transplantation.

5-HT2A receptor-mediated excitation on cerebellar fastigial nucleus neurons and promotion of motor behaviors in rats.

It has long been known that serotonergic afferent inputs are the third largest afferent population in the cerebellum after mossy fibers and climbing fibers. However, the role of serotonergic inputs in cerebellar-mediated motor behaviors is still largely unknown. Here, we show that only 5-HT2A receptors among the 5-HT2 receptor subfamily are expressed and localized in the rat cerebellar fastigial nucleus (FN), one of the ultimate outputs of the spinocerebellum precisely regulating trunk and limb movements. Remarkably, selective activation of 5-HT2A receptors evokes a postsynaptic excitatory effect on FN neurons in a concentration-dependent manner in vitro, which is in accord with the 5-HT-elicited excitation on the same tested neurons. Furthermore, selective 5-HT2A receptor antagonist M100907 concentration-dependently blocks the excitatory effects of 5-HT and TCB-2, a 5-HT2A receptor agonist, on FN neurons. Consequently, microinjection of 5-HT into bilateral FNs significantly promotes rat motor performances on accelerating rota-rod and balance beam and narrows stride width rather than stride length in locomotion gait. All these motor behavioral effects are highly consistent with those of selective activation of 5-HT2A receptors in FNs, and blockage of the component of 5-HT2A receptor-mediated endogenous serotonergic inputs in FNs markedly attenuates these motor performances. All these results demonstrate that postsynaptic 5-HT2A receptors greatly contribute to the 5-HT-mediated excitatory effect on cerebellar FN neurons and promotion of the FN-related motor behaviors, suggesting that serotonergic afferent inputs may actively participate in cerebellar motor control through their direct modulation on the final output of the spinocerebellum.

Histamine excites neurons of the inferior vestibular nucleus in rats by activation of H1 and H2 receptors.

By using brain slice preparations and extracellular recordings, the effect of histamine on spontaneous firing activities of neurons in the inferior vestibular nucleus (IVN), a key structure responsible for integration of vestibular, multisensory, and cerebellar inputs, in rats was investigated. Perfusing slices with histamine (1-10µM) elicited an excitatory response on IVN neurons. The responses were not blocked by low Ca(2+)/high Mg(2+) medium, indicating a direct postsynaptic effect of the amine. Furthermore, the histamine-induced excitation was partially blocked by selective histamine H1 receptor antagonist mepyramine (1µM) and H2 receptor antagonist ranitidine (1µM), respectively. Co-application of mepyramine and ranitidine nearly totally antagonized the histamine-induced excitation. Additionally, both selective H1 receptor agonist 2-pyridylethylamine (30-300µM) and H2 receptor agonist dimaprit (10-100µM) effectively mimicked the excitatory action of histamine on IVN neurons. Moreover, selective H4 antagonist JNJ7777120 (10µM) and agonist VUF8430 (30-300µM) had no effect on IVN neurons. These results demonstrate that histamine excites IVN neurons via postsynaptic H1 and H2 rather than H4 receptors, and suggest that the central histaminergic system actively modulate all four major vestibular nuclei including the IVN and may subsequently influence the vestibular nuclei-related reflexes and functions.

Histamine promotes rat motor performances by activation of H(2) receptors in the cerebellar fastigial nucleus.

The cerebellar fastigial nucleus (FN), together with the interpositus nucleus (IN), constitutes the two final output nuclei of the spinocerebellum and plays an important role in body and limb movements. Previous studies have revealed a direct histaminergic projection from the hypothalamus to the cerebellar nuclei and an excitatory effect of histamine on the IN neurons. However, role of hypothalamic histaminergic projection in the FN has been still little known. Here we show that histamine elicited the FN neurons of rats a concentration-dependent excitatory response in vitro. The histamine-induced excitation on FN neurons was mediated by postsynaptic histamine H2 rather than H1 receptors. In behavioral tests, microinjection of histamine into bilateral FNs remarkably improved motor performances of rats on both accelerating rota-rod and balance beam. Selective H2 receptor antagonist ranitidine considerably declined those motor performances and selective H2 receptor agonist dimaprit mimicked the facilitation effect of histamine on the movements. But selective H1 receptor antagonist triprolidine and agonist 2-pyridylethylamine had no effect. Furthermore, microinjection of histamine into bilateral FNs narrowed stride width of footprint but did not influence wire suspension, whereas microinjection of histamine into bilateral INs increased stride length and promoted suspension. These results demonstrate that histamine enhances rat motor balance and coordination through modulation of both proximal and distal muscles by activation of histamine H2 receptors in the cerebellar FN and IN, and suggest that the hypothalamocerebellar histaminergic projections may modulate the final outputs of the spinocerebellum and participate in the cerebellum-mediated motor control.

A role for orexin in central vestibular motor control.

The absence of orexin results in narcolepsy-cataplexy. While the function of the central orexinergic system in sleep regulation has been well studied, the role of orexin in motor control is largely unknown. Here, we show that orexin-A acts via OX(1) and OX(2) receptors to directly depolarize neurons in the rat lateral vestibular nucleus (LVN), a subcortical motor center, and enhance their sensitivity. A dual ionic mechanism involving both Na+-Ca²+ exchangers and inward rectifier K+ channels underlies these effects. Furthermore, orexin-A regulates central vestibular-mediated posture, motor balance and negative geotaxis. Orexin is critical when an animal is facing a major motor challenge as opposed to during rest and general movements. Therefore, orexin participates not only in sleep and emotion (nonsomatic) but also in motor (somatic) regulation, suggesting that the central orexinergic system plays an important role in somatic-nonsomatic integration. These findings may account for why the absence of orexin results in narcolepsy-cataplexy.