Recent advances of mechanosensitive genes in vascular endothelial cells for the formation and treatment of atherosclerosis.

Atherosclerotic cardiovascular disease and its complications are a high-incidence disease worldwide. Numerous studies have shown that blood flow shear has a huge impact on the function of vascular endothelial cells, and it plays an important role in gene regulation of pro-inflammatory, pro-thrombotic, pro-oxidative stress, and cell permeability. Many important endothelial cell mechanosensitive genes have been discovered, including KLK10, CCN gene family, NRP2, YAP, TAZ, HIF-1α, NF-κB, FOS, JUN, TFEB, KLF2/KLF4, NRF2, and ID1. Some of them have been intensively studied, whereas the relevant regulatory mechanism of other genes remains unclear. Focusing on these mechanosensitive genes will provide new strategies for therapeutic intervention in atherosclerotic vascular disease. Thus, this article reviews the mechanosensitive genes affecting vascular endothelial cells, including classical pathways and some newly screened genes, and summarizes the latest research progress on their roles in the pathogenesis of atherosclerosis to reveal effective therapeutic targets of drugs and provide new insights for anti-atherosclerosis.

Discoveries in Retina Physiology and Disease Biology Using Single-Cell RNA Sequencing.

The retina, a component of the central nervous system, is composed of six distinct neuronal types and various types of glial cells. A technique for single-cell transcriptome analysis called single-cell RNA sequencing (scRNA-seq) can be employed to study the complicated dynamics of several types of retinal cells. It meticulously examines how various cell types express their genes, shedding light on all biological processes. scRNA-seq is an alternative to regular RNA-seq, which cannot identify cellular heterogeneity. Understanding retinal diseases requires research on retinal cell heterogeneity. The identification of novel cell subpopulations can provide information about disease occurrence and progression as well as the specific biological functions of particular cells. We currently have a better understanding of the interactions among the brain, the retina, and its visual pathways thanks to the use of scRNA-seq to examine retinal development and disease pathogenesis. Additionally, this technology offers fresh perspectives on the sensitivity and molecular basis of cell subtypes linked to retinal diseases. Thanks to scRNA-seq technology, we now have a better understanding of the most recent developments and difficulties in retinal development and disorders. We believe that scRNA-seq is an important tool for developing cutting-edge treatments for retinal diseases. This paper presents a systematic review of the history of sRNA-seq technology development and provides an overview of the unique subtypes of retinal cells and the specific gene markers this technology identifies.

Acute exposure of Isopyrazam damages the developed cardiovascular system of zebrafish (Danio rerio).

Isopyrazam (IPZ) is one of the broad-spectrum succinate dehydrogenase inhibitor fungicides (SDHIs). Although the potential bio-toxicity of SDHIs has been reported hourly, the specific effects focused on the cardiovascular system have remained unclear and piecemeal. Thus, we chose IPZ as a representative to observe the cardiovascular toxicity of SDHIs in zebrafish. Two types of transgenic zebrafish, Tg (cmlc2:GFP) and Tg (flk1:GFP) were used in this study. Healthy embryos at 6 hpf were exposed to IPZ solutions. The statistical data including survival rate, hatching rate, malformed rate, and morphological and functional parameters of the cardiovascular system at 48 hpf and 72 hpf demonstrated that IPZ could cause abnormalities and cardiovascular defects such as spinal curvature, dysmotility, pericardial edema, pericardial hemorrhage, and slowed heart rate, etc. At the same time, the activity of enzymes related to oxidative stress was altered with IPZ. Our results revealed that IPZ-induced cardiovascular toxicity and oxidative stress might be one of the underlying toxic mechanisms.

Noncanonical protease-activated receptor 1 regulates lymphatic differentiation in zebrafish.

The differentiation of lymphatic progenitors is a crucial step in lymphangiogenesis. However, its underlying mechanism remains unclear. Here, we found that noncanonical protease-activated receptor 1 (par1) regulates the differentiation of lymphatic progenitors in zebrafish embryos. Loss of par1 function impaired lymphatic differentiation by downregulating prox1a expression in parachordal lymphangioblasts and caused compromised thoracic duct formation in zebrafish. Meanwhile, the G protein gnai2a, a par1 downstream effector, was selectively required for lymphatic development in zebrafish, and its mutation mimicked the lymphatic phenotype observed in par1 mutants. Interestingly, mmp13, but not thrombin, was required for lymphatic development in zebrafish. Furthermore, analyses of genetic interactions confirmed that mmp13b serves as a par1 upstream protease to regulate lymphatic development in zebrafish embryos. Mechanistically, par1 promotes flt4 expression and phospho-Erk1/2 activity in the posterior cardinal vein. Taken together, our findings highlight a function of par1 in the regulation of lymphatic differentiation in zebrafish embryos.

The blood flow-klf6a-tagln2 axis drives vessel pruning in zebrafish by regulating endothelial cell rearrangement and actin cytoskeleton dynamics.

Recent studies have focused on capillary pruning in various organs and species. However, the way in which large-diameter vessels are pruned remains unclear. Here we show that pruning of the zebrafish caudal vein (CV) from ventral capillaries of the CV plexus in different transgenic embryos is driven by endothelial cell (EC) rearrangement, which involves EC nucleus migration, junction remodeling, and actin cytoskeleton remodeling. Further observation reveals a growing difference in blood flow velocity between the two vessels in CV pruning in zebrafish embryos. With this model, we identify the critical role of Kruppel-like factor 6a (klf6a) in CV pruning. Disruption of klf6a functioning impairs CV pruning in zebrafish. klf6a is required for EC nucleus migration, junction remodeling, and actin cytoskeleton dynamics in zebrafish embryos. Moreover, actin-related protein transgelin 2 (tagln2) is a direct downstream target of klf6a in CV pruning in zebrafish embryos. Together these results demonstrate that the klf6a-tagln2 axis regulates CV pruning by promoting EC rearrangement.

Engineered bioresponsive nanotherapeutics: recent advances in the treatment of atherosclerosis and ischemic-related disease.

Biological stimuli that are present during the pathogenesis of disease have gained considerable interest as a critical element for the design of smart drug delivery systems. Recently, the utilization of biological stimuli-responsive (bioresponsive) nanotheranostic agents to treat atherosclerosis and ischemic-related diseases has demonstrated significant outcomes in preclinical studies. Those diseases share similar hallmarks, including high levels of endogenous reactive oxygen species (ROS), low pH, and high enzyme activity. Interestingly, other relevant biological stimuli such as shear stress, cholesterol, and glutathione have recently been explored as internal stimuli to trigger drug release and some particular actions. In addition, a number of strategies can be proposed to enhance their targeting efficiency, diagnostic properties, and efficacy rate. This review discusses recent advancements in the preclinical studies of bioresponsive nanotherapeutics as diagnostic and therapeutic agents against atherosclerosis and ischemic-related diseases as well as some potential strategies to overcome the current limitations.

Developmental neurotoxicity of antimony (Sb) in the early life stages of zebrafish.

Accumulating studies have revealed the toxicity of antimony (Sb) to soil-dwelling and aquatic organisms at the individual level. However, little is known about the neurotoxic effects of antimony and its underlying mechanisms. To assess this issue, we investigated the neurotoxicity of antimony (0, 200, 400, 600 and 800 mg/L) in zebrafish embryos. After exposure, zebrafish embryos showed abnormal phenotypes such as a shortened body length, morphological malformations, and weakened heart function. Behavioral experiments indicated that antimony caused neurotoxicity in zebrafish embryos, manifested in a decreased spontaneous movement frequency, delayed response to touch, and reduced movement distance. We also showed that antimony caused a decrease in acetylcholinesterase (AChE) levels in zebrafish embryos, along with decreased expression of neurofunctional markers such as gfap, nestin, mbp, and shha. Additionally, antimony significantly increased reactive oxygen species levels and significantly reduced glutathione (GSH) and superoxide dismutase (SOD) activity. In summary, our findings indicated that antimony can induce developmental toxicity and neurotoxicity in zebrafish embryos by affecting neurotransmitter systems and oxidative stress, thus altering behavior. These outcomes will advance our understanding of antimony-induced neurotoxicity, environmental problems, and health hazards.

Cadmium-induced dysfunction of the blood-brain barrier depends on ROS-mediated inhibition of PTPase activity in zebrafish.

Increasing evidence has demonstrated that cadmium accumulation in the blood increases the risk of neurological diseases. However, how cadmium breaks through the blood-brain barrier (BBB) and is transferred from the blood circulation into the central nervous system is still unclear. In this study, we examined the toxic effect of cadmium chloride (CdCl2) on the development and function of BBB in zebrafish. CdCl2 exposure induced cerebral hemorrhage, increased BBB permeability and promoted abnormal vascular formation by promoting VEGF production in zebrafish brain. Furthermore, in vivo and in vitro experiments showed that CdCl2 altered cell-cell junctional morphology by disrupting the proper localization of VE-cadherin and ZO-1. The potential mechanism involved in the inhibition of protein tyrosine phosphatase (PTPase) mediated by cadmium-induced ROS was confirmed with diphenylene iodonium (DPI), a ROS production inhibitor. Together, these data indicate that BBB is a critical target of cadmium toxicity and provide in vivo etiological evidence of cadmium-induced neurovascular disease in a zebrafish BBB model.

FoxF1 is Required for Ciliogenesis and Distribution of Sonic Hedgehog Signaling Components in Cilium.

BACKGROUND: In vertebrates, cilium is crucial for Hedgehog signaling transduction. Forkhead box transcriptional factor FoxF1 is reported to be associated with Sonic Hedgehog (Shh) signaling in many cases. However, the role of FoxF1 in cilium remains unknown. Here, we showed an essential role of FoxF1 in the regulation of ciliogenesis and in the distribution of Shh signaling components in cilium. METHODS: NIH/3T3 cells were serum starved for 24h to induce cilium. Meanwhile, shRNA was used to knockdown the FoxF1 expression in the cells and CRISPR/Cas9 was used to generate the FoxF1 zebrafish mutant. The mRNA and protein expression of indicated genes were detected by the qRT-PCR and western blot, respectively. Immunofluorescence staining was performed to detect the cilium and Shh components distribution. RESULTS: FoxF1 knockdown decreased the cilium length in NIH/3T3 cells. Meanwhile, the disruption of FoxF1 function inhibited the expression of cilium-related genes and caused an abnormal distribution of Shh components in the cilium. Furthermore, homozygous FoxF1 mutants exhibited defective development of pronephric cilium in early zebrafish embryos. CONCLUSION: Together, our data illustrated that FoxF1 is required for ciliogenesis in vitro and in vivo and for the proper localization of Shh signaling components in cilium.

Effect of simulated microgravity induced PI3K-nos2b signalling on zebrafish cardiovascular plexus network formation.

Local abnormal angiogenesis and cardiovascular system reorganization have been observed in embryos exposed to a simulated microgravity (SM) environment. In this study, changes in key molecular signals and pathways in cardiovascular development have been investigated under microgravity conditions. In particular, the caudal vein plexus (CVP) network, formed by sprouting angiogenesis has been chosen. Zebrafish embryos were exposed to SM using a ground-based microgravity bioreactor for 24 and 36 h. The SM was observed to have no effect on the zebrafish length, tail width and incubation time whereas it was observed to significantly reduce the heart rate frequency and to promote abnormal development of the CVP network in the embryos. Nitric oxide (NO) content demonstrated that the total proteins in zebrafish embryos were significantly higher in SM than in the control group grown under normal conditions. It was then preliminarily determined how NO signals were involved in SM regulated zebrafish CVP network formation. nos2b MO was injected and CVP network evolution was observed in 36 h post fertilization (hpf) under SM condition. The results showed that the CVP network formation was considerably decreased in the nos2b MO treated group. However, this inhibition of the CVP network development was not observed in control MO group, indicating that nos2b is involved in the SM-regulated vascular development process in zebrafish. Moreover, specific phosphoinositide 3-kinase (PI3K) inhibitors such as LY294002 were also tested on zebrafish embryos under SM condition. This treatment significantly inhibited the formation of zebrafish CVP network. Furthermore, overexpression of nos2b partly rescued the LY294002-caused CVP network failure. Therefore, it can be concluded that SM affects zebrafish CVP network remodeling by enhancing angiogenesis. Additionally, the PI3K-nos2b signaling pathway is involved in this process.

Effect of intraplaque angiogenesis to atherosclerotic rupture-prone plaque induced by high shear stress in rabbit model.

Atherosclerotic prone-rupture plaque is mainly localized in the region of the entrance to the stenosis with high shear stress and the reasons are largely unknown. Our hypothesis is that such a distribution of cells in atherosclerotic plaque may depend on the angiogenesis. Silastic collars induced regions of high shear stress (20.68 ± 5.27 dynes/cm2) in the upstream flow and low shear stress (12.25 ± 1.28 dynes/cm2) in the downstream flow in carotid arteries. Compared with the low shear stress region, plaques in the high shear stress region showed more intraplaque haemorrhaging, less collagen and higher apoptotic rates of vascular smooth muscle cells; endothelial cells (ECs) in the high shear stress region were characterized with integrity and high endothelial nitric oxide synthase (eNOS) expression (1570.3 ± 345.5% vs 172.9 ± 49.9%). The number of intraplaque microvessels is very high in the high shear stress region (15 ± 1.8 n/mm2 vs 3.5 ± 0.4 n/mm2), and the microvessels in the plaque show ECs were abnormal, with membrane blebs, intracytoplasmic vacuoles and leukocyte infiltration. Our current study reveals that the integrity of the endothelium and the vulnerability of atherosclerotic plaques are simultaneously localized in high shear stress regions, and we provide evidence for the first time that microvessels in the intraplaque maybe responsible for rupture-prone plaque formation in the high shear stress region.

Penetration of blood-brain barrier and antitumor activity and nerve repair in glioma by doxorubicin-loaded monosialoganglioside micelles system.

For the treatment of glioma and other central nervous system diseases, one of the biggest challenges is that most therapeutic drugs cannot be delivered to the brain tumor tissue due to the blood-brain barrier (BBB). The goal of this study was to construct a nanodelivery vehicle system with capabilities to overcome the BBB for central nervous system administration. Doxorubicin as a model drug encapsulated in ganglioside GM1 micelles was able to achieve up to 9.33% loading efficiency and 97.05% encapsulation efficiency by orthogonal experimental design. The in vitro study demonstrated a slow and sustainable drug release in physiological conditions. In the cellular uptake studies, mixed micelles could effectively transport into both human umbilical vein endothelial cells and C6 cells. Furthermore, biodistribution imaging of mice showed that the DiR/GM1 mixed micelles were accumulated sustainably and distributed centrally in the brain. Experiments on zebrafish confirmed that drug-loaded GM1 micelles can overcome the BBB and enter the brain. Among all the treatment groups, the median survival time of C6-bearing rats after administering DOX/GM1 micelles was significantly prolonged. In conclusion, the ganglioside nanomicelles developed in this work can not only penetrate BBB effectively but also repair nerves and kill tumor cells at the same time.

Standardization of a well-controlled in vivo mouse model of thrombus formation induced by mechanical injury.

OBJECTIVE: Vascular plug formation by mechanical injury that exposes abundant extracellular matrix is an ideal model to mimic thrombus formation. The objective of this study was to standardize our previously established in vivo mouse model of thrombus formation induced by mechanical injury. RESULTS: The mechanical injury was exerted by pinching the abdominal aorta with hemostatic forceps for either 15 (moderate injury) or 60 (severe injury) seconds. Thrombus formation was monitored for 20min in real time using a fluorescent microscope coupled to a CCD camera. In the moderate injury, thrombus formation peaked at approximately 1min after injury and resolved within 3min, with the mean AUC (area under the curve) of 165.2±17.29mm(2), whereas a larger thrombus was observed upon the severe injury, with the mean AUC of 600.5±37.77mm(2). Using scanning electron microscopy and HE staining, a complete deformation of the endothelium in the moderate injury model and the exposure of the media in the severe injury model were observed. The model was also evaluate for its application on the effects of antithrombotic drugs targeting GP IIb-IIIa (eptifibatide), ADP receptor P2Y1 (MRS2500) and P2Y12 (clopidogrel), and thrombin (hirudin) on thrombus formation. CONCLUSIONS: We have improved a vascular injury model with optimal reproducibility and feasibility that allows evaluating the effect of anti-thrombotic drugs on thrombus formation in vivo.

An in vitro study on the biocompatibility of WE magnesium alloys.

Magnesium alloys are being actively studied for intravascular stent applications because of their good mechanical strength and biocompatibility. To rule out the high allergenicity of nickel and neurotoxicity of aluminum element, four kinds of WE magnesium alloys (where "W" represents the metallic element Y and "E" represents mixed rare earth (RE) elements; Y: 2.5, 5.0, 6.5, and 7.5 wt %; Nd: 1.0, 2.6, 2.5, and 4.2 wt %; Zr: 0.8 wt %) were chosen for in vitro investigation of their biocompatibility using cell culture. The results showed that, with the increase of rare earth elements in WE magnesium alloys, fibrinogen adsorption decreased and coagulation function was improved. It was also found that WE magnesium alloys promoted the adhesion of endothelial cells. With the increase of adhesion time, adhered cell numbers increased gradually. With 25% extracts, all the WE alloys promoted cell migration, while 100% extracts were not conducive to cell migration. Based on the above results, WE magnesium alloys 5.0WE (5.0Y-2.6Nd-0.8Zr) and 6.5WE (6.5Y-2.5Nd-0.8Zr) have better biocompatibility as compared with that with 2.5WE (2.5Y-1.0Nd-0.8Zr) and 7.5WE (7.5Y-4.2Nd-0.8Zr), and could be as the promising candidate materials for medical stent applications.

[Effect of horizontal rotary culture on zebrafish vascular development].

With the development of space life science, a study on the influence of microgravity on organism has been an increasingly concerned topic. Lots of studies indicate that microgravity plays an important role in the early development of embryos. The vascular system as the first-function system of embryos provides an interesting topic for many researchers. However, those studies were mostly carried out in vitro by rotary cell culture system (RCCS), while few experiments were done in vivo. Using zebrafish as a model, this research investigated the effects of horizontal rotary culture on the vascular development in vivo. Zebrafish embryos at 24 hpf (hour post-fertilization) were selected and divided into two groups. One group was cultured by the shaker, and the other was cultured normally as the control. After 12 h, all the embryos were collected and detected. The phenotype of zebrafish was observed by stereo microscope. Then, the expression of vascular specific expression factor, flk1, flt4, and ephrinB2 was compared by RT-PCR, qPCR, and in situ hybridization, respectively. Cell apoptosis and proliferation in situ were observed using TUNEL assay and bromodeoxyuridine incorporation. The results demonstrated that horizontal rotary culture at 90 r/min decreased the hatching of embryos (10.3±0.41 vs. 0.0, P<0.05), accelerate the heart rate (223.5±2.32 vs. 185.0±3.23, P<0.05) and increased the content of melanin in zebrafish significantly. At the same time, we found some differences in the vascular system of zebrafish after horizontal rotary culture which caused a down regulation of flk1, flt4, and ephrinB2. On the other hand, horizontal rotary culture accelerated the apoptosis of cells in zebrafish, but showed no significance in proliferation. In conclusion, horizontal rotary culture has a significant influence on the vascular development in zebrafish.

In vitro and in vivo investigations on the effects of low-density lipoprotein concentration polarization and haemodynamics on atherosclerotic localization in rabbit and zebrafish.

Atherosclerosis (AS) commonly occurs in the regions of the arterial tree with haemodynamic peculiarities, including local flow field disturbances, and formation of swirling flow and vortices. The aim of our study was to confirm low-density lipoprotein (LDL) concentration polarization in the vascular system in vitro and in vivo, and investigate the effects of LDL concentration polarization and flow field alterations on atherosclerotic localization. Red fluorescent LDL was injected into optically transparent Flk1: GFP zebrafish embryos, and the LDL distribution in the vascular lumen was investigated in vivo using laser scanning confocal microscopy. LDL concentration at the vascular luminal surface was found to be higher than that in the bulk. The flow field conditions in blood vessel segments were simulated and measured, and obvious flow field disturbances were found in the regions of vascular geometry change. The LDL concentration at the luminal surface of bifurcation was significantly higher than that in the straight segment, possibly owing to the atherogenic effect of disturbed flow. Additionally, a stenosis model of rabbit carotid arteries was generated. Atherosclerotic plaques were found to have occurred in the stenosis group and were more severe in the stenosis group on a high-fat diet. Our findings provide the first ever definite proof that LDL concentration polarization occurs in the vascular system in vivo. Both lipoprotein concentration polarization and flow field changes are involved in the infiltration/accumulation of atherogenic lipids within the location of arterial luminal surface and promote the development of AS.