Genetically Encoded Fluorescence Resonance Energy Transfer Biosensor for Live-Cell Visualization of Lamin A Phosphorylation at Serine 22.

Extensive phosphorylation at serine 22 (pSer22) on lamin A is the hallmark of cell mitosis, which contributes to the breakdown of nuclear envelope. In the interphase, pSer22 lamin A exists in low abundance and is involved in mechanotransduction, virus infection, and gene expression. Numerous evidences emerge to support lamin A regulation on cell function and fate by phosphorylation. However, live-cell imaging tools for visualizing the dynamics of pSer22 lamin A are yet to be established. Herein, we developed a novel lamin A phosphorylation sensor (LAPS) based on fluorescence resonance energy transfer (FRET) with high sensitivity and specificity. We observed the dynamic lamin A phosphorylation during the cell cycle progression in single living cells: the increase of pSer22 modification when cells entered the mitosis and recovered upon the mitosis exit. Our biosensor also showed the gradual reduction of pSer22 modification during cell adhesion and in response to hypotonic environment. By applying LAPS, we captured the propagation of pSer22 modification from inside to outside of the inner nuclear membrane, which further led to the breakdown of nuclear envelope. Meanwhile, we found the synchronous phosphorylation of pSer22 lamin A and H3S10ph at mitosis entry. Inhibition of Aurora B, the responsible kinase for H3S10ph, could shorten the mitotic period without obvious effect on the pSer22 modification level of lamin A. Thus, LAPS allows the spatiotemporal visualization of the lamin A pSer22, which will be useful for elucidating the molecular mechanisms underlying cell mitosis and mechanoresponsive processes.

The interplay between histone modifications and nuclear lamina in genome regulation.

Gene expression is regulated by chromatin architecture and epigenetic remodeling in cell homeostasis and pathologies. Histone modifications act as the key factors to modulate the chromatin accessibility. Different histone modifications are strongly associated with the localization of chromatin. Heterochromatin primarily localizes at the nuclear periphery, where it interacts with lamina proteins to suppress gene expression. In this review, we summarize the potential bridges that have regulatory functions of histone modifications in chromatin organization and transcriptional regulation at the nuclear periphery. We use lamina-associated domains (LADs) as examples to elucidate the biological roles of the interactions between histone modifications and nuclear lamina in cell differentiation and development. In the end, we highlight the technologies that are currently used to identify and visualize histone modifications and LADs, which could provide spatiotemporal information for understanding their regulatory functions in gene expression and discovering new targets for diseases.

Unravelling the antioxidant behaviour of self-assembly ß-Sheet in silk fibroin.

Local oxidative stress in diseases or injury severely hinders cell homeostasis and organ regeneration. Antioxidant therapy is an effective strategy for oxidative stress treatment. Biomaterials with good biocompatibility and reactive oxygen species (ROS) scavenging ability are good choices for antioxidant therapeutics. However, there are few natural biomaterials that are identified with both biocompatibility and strong antioxidant activity. Here, we show, for the first time, that silk fibroin (SF) is a strong antioxidant, which can eliminate ROS in both cells and zebrafish. We further demonstrate that the ß-sheet structures turn into a random coiled structure when SF is treated with hydrogen peroxide. The content of ß-sheet structures can be increased by heating, thus enhancing the antioxidation properties of SF. Therefore, SF can serve as a good antioxidant biomaterial for therapeutics, and its ß-sheet structure-based antioxidation mechanism provides a novel theoretical basis, which could be a new cue for more antioxidant biomaterial discovery and identification.

Coupled single-cell and bulk RNA-seq analysis reveals the engulfment role of endothelial cells in atherosclerosis.

The clearance of apoptotic cell debris, containing professional phagocytosis and non-professional phagocytosis, is essential for maintaining the homeostasis of healthy tissues. Here, we discovered that endothelial cells could engulf apoptotic cell debris in atherosclerotic plaque. Single-cell RNA sequencing (RNA-seq) has revealed a unique endothelial cell subpopulation in atherosclerosis, which was strongly associated with vascular injury-related pathways. Moreover, integrated analysis of three vascular injury-related RNA-seq datasets showed that the expression of scavenger receptor class B type 1 (SR-B1) was up-regulated and specifically enriched in the phagocytosis pathway under vascular injury circumstances. Single-cell RNA-seq and bulk RNA-seq indicate that SR-B1 was highly expressed in a unique endothelial cell subpopulation of mouse aorta and strongly associated with the reorganization of cellular adherent junctions and cytoskeleton which were necessary for phagocytosis. Furthermore, SR-B1 was strongly required for endothelial cells to engulf apoptotic cell debris in atherosclerotic plaque of both mouse and human aorta. Overall, this study demonstrated that apoptotic cell debris could be engulfed by endothelial cells through SR-B1 and associated with the reorganization of cellular adherent junctions and cytoskeleton.

Stress granules in atherosclerosis: Insights and therapeutic opportunities.

Atherosclerosis, a complex inflammatory and metabolic disorder, is the underlying cause of several life-threatening cardiovascular diseases. Stress granules (SG) are biomolecular condensates composed of proteins and mRNA that form in response to stress. Recent studies suggest a potential link between SG and atherosclerosis development. However, there remain gaps in understanding SG role in atherosclerosis development. Here we provide a thorough analysis of the role of SG in atherosclerosis, covering cellular stresses stimulation, core components, and regulatory genes in SG formation. Furthermore, we explore atherosclerosis induced factors such as inflammation, low or oscillatory shear stress (OSS), and oxidative stress (OS) may impact SG formation and then the development of atherosclerotic lesions. We have assessed how changes in SG dynamics impact pro-atherogenic processes like endothelial dysfunction, lipid metabolism, and immune cell recruitment in atherosclerosis. In summary, this review emphasizes the complex interplay between SG and atherosclerosis that could open innovative directions for targeted therapeutic strategies in preventing or treating atherosclerotic cardiovascular diseases.

Silk fibroin promotes H3K9me3 expression and chromatin reorganization to regulate endothelial cell proliferation.

Silk fibroin (SF), which is extensively utilized in tissue engineering and vascular grafts for enhancing vascular regeneration, has not been thoroughly investigated for its epigenetic effects on endothelial cells (EC). This study employed RNA sequencing analysis to evaluate the activation of histone modification regulatory genes in EC treated with SF. Subsequent investigations revealed elevated H3K9me3 levels in SF-treated EC, as evidenced by immunofluorescence and western blot analysis. The study utilized H2B-eGFP endothelial cells to demonstrate that SF treatment results in the accumulation of H2B-marked chromatin in the nuclear inner cavities of EC. Inhibition of H3K9me3 levels by a histone deacetylase inhibitor TSA decreased cell proliferation. Furthermore, the activation of the MAPK signaling pathway using chromium picolinate decreased the proliferative activity and H3K9me3 level in SF-treated EC. SF also appeared to enhance cell growth and proliferation by modulating the H3K9me3 level and reorganizing chromatin, particularly after oxidative stress induced by H2O2 treatment. In summary, these findings indicate that SF promotes EC proliferation by increasing the H3K9me3 level even under stress conditions.

Universal cell membrane camouflaged nano-prodrugs with right-side-out orientation adapting for positive pathological vascular remodeling in atherosclerosis.

A right-side-out orientated self-assembly of cell membrane-camouflaged nanotherapeutics is crucial for ensuring their biological functionality inherited from the source cells. In this study, a universal and spontaneous right-side-out coupling-driven ROS-responsive nanotherapeutic approach, based on the intrinsic affinity between phosphatidylserine (PS) on the inner leaflet and PS-targeted peptide modified nanoparticles, has been developed to target foam cells in atherosclerotic plaques. Considering the increased osteopontin (OPN) secretion from foam cells in plaques, a bioengineered cell membrane (OEM) with an overexpression of integrin α9ß1 is integrated with ROS-cleavable prodrugs, OEM-coated ETBNPs (OEM-ETBNPs), to enhance targeted drug delivery and on-demand drug release in the local lesion of atherosclerosis. Both in vitro and in vivo experimental results confirm that OEM-ETBNPs are able to inhibit cellular lipid uptake and simultaneously promote intracellular lipid efflux, regulating the positive cellular phenotypic conversion. This finding offers a versatile platform for the biomedical applications of universal cell membrane camouflaging biomimetic nanotechnology.

Oscillatory shear stress-induced downregulation of TET1s injures vascular endothelial planar cell polarity by suppression of actin polymerization.

Vascular endothelial polarity induced by blood flow plays crucial roles in the development of atherosclerosis. Loss of endothelial polarity leads to an increase in permeability and leukocyte recruitment, which are crucial hallmarks of atherosclerotic initiation. Endothelial cells exhibit a morphological adaptation to hemodynamic shear stress and possesses planar cell polarity to the direction of blood flow. However, the mechanism of how hemodynamic shear stress regulates endothelial planar cell polarity has not been firmly established. Here, we found that TET1s, a short isoform of Tet methylcytosine dioxygenase 1, was a mediator in the regulation of the planar cell polarity in endothelial cells in response to hemodynamic shear stress. In the process, low expression of TET1s induced by oscillatory shear stress led to the endothelial planar polarity damage through inhibition of F-actin polymerization. TET1s can regulate demethylation level of the sFRP-1 promoter to alter the expression of sFRP-1, which affects the interaction of sFRP-1/Fzd4 and F-actin polymerization. Our study revealed the mechanism of how TET1s mediates endothelial planar cell polarity in response to hemodynamic shear stress and provides a new insight for the prevention of atherosclerosis.

Shear stress regulation of nanoparticle uptake in vascular endothelial cells.

Nanoparticles (NPs) hold tremendous targeting potential in cardiovascular disease and regenerative medicine, and exciting clinical applications are coming into light. Vascular endothelial cells (ECs) exposure to different magnitudes and patterns of shear stress (SS) generated by blood flow could engulf NPs in the blood. However, an unclear understanding of the role of SS on NP uptake is hindering the progress in improving the targeting of NP therapies. Here, the temporal and spatial distribution of SS in vascular ECs and the effect of different SS on NP uptake in ECs are highlighted. The mechanism of SS affecting NP uptake through regulating the cellular ROS level, endothelial glycocalyx and membrane fluidity is summarized, and the molecules containing clathrin and caveolin in the engulfment process are elucidated. SS targeting NPs are expected to overcome the current bottlenecks and change the field of targeting nanomedicine. This assessment on how SS affects the cell uptake of NPs and the marginalization of NPs in blood vessels could guide future research in cell biology and vascular targeting drugs.

Desulfovibrio desulfuricans aggravates atherosclerosis by enhancing intestinal permeability and endothelial TLR4/NF-κB pathway in Apoe -/- mice.

It is increasingly aware that gut microbiota is closely associated with atherosclerosis. However, which and how specific gut bacteria regulate the progression of atherosclerosis is still poorly understood. In this study, modified linear discriminant analysis was performed in comparing the gut microbiota structures of atherosclerotic and non-atherosclerotic mice, and Desulfovibrio desulfuricans (D. desulfuricans) was found to be associated with atherosclerosis. D. desulfuricans-treated Apoe -/- mice showed significantly aggravated atherosclerosis. The proatherogenic effect of D. desulfuricans was attributed to its ability to increase intestinal permeability and subsequent raise in the transit of lipopolysaccharide (LPS) from the intestine to the bloodstream. Excessive LPS in the blood can elicit local and systemic inflammation and activate Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling of endothelial cells. TAK-242, a specific inhibitor of TLR4, can ameliorate the development of D. desulfuricans-induced atherosclerosis by blocking the LPS-induced activation of TLR4/NF-κB signaling.

Spontaneously Right-Side-Out-Orientated Coupling-Driven ROS-Sensitive Nanoparticles on Cell Membrane Inner Leaflet for Efficient Renovation in Vascular Endothelial Injury.

Biomimetic cell membrane camouflaged technology has drawn extensive attention as a feasible and efficient way to realize the biological functions of nanoparticles from the parent cells. As the burgeoning nanotherapeutic, the right-side-out orientation self-assembly and pathological dependent "on-demand" cargo release of cell membrane camouflaged nanocarriers remarkably limit further development for practical applications. In the present study, a spontaneously right-side-out-orientated coupling-driven ROS-sensitive nanotherapeutic has been constructed for target endothelial cells (ECs) repair through the synergistic effects of spontaneously right-side-out-orientated camouflaging. This condition results from the specific affinity between the intracellular domain of key transmembrane receptors band 3 on cell membrane inner leaflet and the corresponding P4.2 peptide-modified nanoparticles without the additional coextrusion. The "on-demand" cargo release results from the pathological ROS-cleavable prodrug. Particularly, the red blood cell camouflaged nanotherapeutics (RBC-LVTNPs) can enhance target drug delivery through low oscillatory shear stress (LSS) blood flow in the injured ECs lesion. Both in vitro and in vivo results collectively confirm that RBC-LVTNPs can restore the damaged ECs and function with the recovered vascular permeability and low inflammation microenvironment. The findings provide a powerful and universal approach for developing the biomimetic cell membrane camouflaged nanotechnology.

Prodrug Integrated Envelope on Probiotics to Enhance Target Therapy for Ulcerative Colitis.

Ulcerative colitis (UC), affecting millions of patients worldwide, is associated with disorders of the gut microbiota. Probiotics-based therapy positively regulating the community structure of gut microbiota is regarded as an efficient intervention for UC. However, oral probiotics delivery is restricted by limited bioactivity, short retention time, complex pathological condition, and single therapeutic efficacy. Here, a bioengineered probiotic decorated with a multifunctional prodrug coating is constructed to ameliorate the aforementioned shortcomings. The results of UC mice induced by dextran sulfate sodium demonstrate that the intrinsic features of the fabricated coating integrate gut microbes protection, colon-targeted drug release, prolonged drug retention, and inflammation regulation. In parallel, the probiotics Lactobacillus rhamnosus GG (LGG) could regulate the composition of the gut microbiota and improve epithelial barrier function, thereby synergistically ameliorating UC. These results provide ample shreds of evidence of the therapeutic effect on UC, therefore, demonstrate a great promise as the potential therapeutic strategy for UC treatment.

G3BP2 regulates oscillatory shear stress-induced endothelial dysfunction.

GTPase-activating SH3 domain-binding protein 2 (G3BP2) is a mediator that responds to environmental stresses through stress granule formation and is involved in the progression of chronic diseases. However, no studies have examined the contribution of G3BP2 in the oscillatory shear stress (OSS)-induced endothelial dysfunction. Here we assessed the effects of G3BP2 in endothelial cells (ECs) function and investigated the underlying mechanism. Using shear stress apparatus and partial ligation model, we identified that stress granule-related genes in ECs could be induced by OSS with RNA-seq, and then confirmed that G3BP2 was highly and specifically expressed in athero-susceptible endothelia in the OSS regions. G3bp2 -/- Apoe -/- mice had significantly decreased atherosclerotic lesions associated with deficiency of G3BP2 in protecting endothelial barrier function, decreasing monocyte adhesion to ECs and inhibiting the proinflammatory cytokine levels. Furthermore, loss of G3BP2 diminished OSS-induced inflammation in ECs by increasing YAP nucleocytoplasmic shuttling and phosphorylation. These data demonstrate that G3BP2 is a critical OSS regulated gene in regulating ECs function and that G3BP2 inhibition in ECs is a promising atheroprotective therapeutic strategy.

Biomechanical regulation of planar cell polarity in endothelial cells.

Cell polarity refers to the uneven distribution of certain cytoplasmic components in a cell with a spatial order. The planar cell polarity (PCP), the cell aligns perpendicular to the polar plane, in endothelial cells (ECs) has become a research hot spot. The planar polarity of ECs has a positive significance on the regulation of cardiovascular dysfunction, pathological angiogenesis, and ischemic stroke. The endothelial polarity is stimulated and regulated by biomechanical force. Mechanical stimuli promote endothelial polarization and make ECs produce PCP to maintain the normal physiological and biochemical functions. Here, we overview recent advances in understanding the interplay and mechanism between PCP and ECs function involved in mechanical forces, with a focus on PCP signaling pathways and organelles in regulating the polarity of ECs. And then showed the related diseases caused by ECs polarity dysfunction. This study provides new ideas and therapeutic targets for the treatment of endothelial PCP-related diseases.

Temporal-spatial low shear stress induces heterogenous distribution of hematopoietic stem cell budding in zebrafish.

Hematopoietic stem/progenitor cells (HSPCs) originate from endothelial cells (ECs) localized on the ventral side of the dorsal aorta (DA), and hemodynamic parameters may suffer sharp changes in DA at HSPCs development stage for intersegmental vessel formation. However, the temporal-spatial shear stress parameters and biomechanics mechanisms of HSPC budding remain unknown. Here, we found that the hematopoietic endothelium (HE) in the aorta-gonad-mesonephros was heterogeneous; that is, HEs were mainly distributed at the ventral side of the vascular bifurcation in zebrafish embryos, which was found to show low shear stress (LSS) through numerical simulation analysis. Furthermore, HSPCs localized in the posterior somite of aorta-gonad-mesonephros with slow velocity. On the temporal scale, there was a slow velocity and LSS during HE budding from 36 h post-fertilization and decreased shear stress with drug expanded HSPC numbers. Mechanistically, matrix metalloproteinase (MMP) expression and macrophage chemotaxis were significantly increased in HEs by RNA-seq. After treatment with an MMP13 inhibitor, HSPCs were significantly reduced in both the aorta-gonad-mesonephros and caudal hematopoietic tissue in embryos. Our results show that HSPC budding is heterogeneous, and the mechanism is that physiological LSS controls the emergence of HSPCs by promoting the accumulation of macrophages and subsequent MMP expression.

trans-2-Enoyl-CoA Reductase Tecr-Driven Lipid Metabolism in Endothelial Cells Protects against Transcytosis to Maintain Blood-Brain Barrier Homeostasis.

The transport and metabolism of lipids in cerebrovascular endothelial cells (ECs) have been hypothesized to regulate blood-brain barrier (BBB) maturation and homeostasis. Long-chain polyunsaturated fatty acids (LCPUFAs) as the important lipids components of cell membranes are essential for the development and function of BBB, but the direct links of lipid metabolism and ECs barrier function remain to be established. Here, we comprehensively characterize the transcriptomic phenotype of developmental cerebrovascular ECs in single-cell resolution and firstly find that trans-2-enoyl-CoA reductase (Tecr), a very-long-chain fatty acid synthesis, is highly expressed during barriergenesis and decreased after BBB maturation. EC-specific knockout of Tecr compromises angiogenesis due to delayed vascular sprouting. Importantly, EC-specific deletion of Tecr loss restrictive quality of vascular permeability from neonatal stages to adulthood, with high levels of transcytosis, but maintains the vascular tight junctions. Moreover, lipidomic analysis shows that the expression of Tecr in ECs is associated with the containing of omega-3 fatty acids, which directly suppresses caveolae vesicles formation. These results reveal a protective role for Tecr in BBB integrity and suggest that Tecr as a novel therapeutic target in the central nervous system (CNS) diseases associated with BBB dysfunction.

TET1s deficiency exacerbates oscillatory shear flow-induced atherosclerosis.

Background: TET1 has been implicated in regulating inflammation and cardiovascular disease, but a newly discovered short isoform of TET1 (termed TET1s) exhibits higher expression in adult tissues than full-length TET1. However, the precise role of TET1 in cardiovascular disease remains undefined. Methods and Results: Based on TET1-/- knockout mice (with deletion of both TET1 and TET1s ) and TET1cs/cs mice (with deletion of only TET1), we found that TET1s deletion in TET1-/- mice resulted in more serious atherosclerotic lesions in the whole aorta than TET1cs/cs in the ApoE-/- background mice fed a high-fat diet. Atherosclerotic lesions with Oil red staining were dramatically localized in the aortic arch, abdominal aorta and ligated LCA, where they were exposed to OSS. Furthermore, the OSS-induced depression of TET1s in vitro and in vivo increased inflammatory cell and red blood cell infiltration into the subendothelial layer by impairing the vascular intimal barrier. TET1s upregulation enhanced vascular endothelial barrier function by increasing gap protein connexin 40 (CX40) expression as measured by RNA-seq and was confirmed by CX40 knockdown. TET1s interaction with Sin3a increased the global and CX40 promoter histone H3K27 acetylation levels, but not DNA methylation, to induce CX40 expression. Conclusions: These data demonstrate the unexpected discovery that laminar shear stress induces TET1s expression to protect the vascular endothelial barrier by increasing CX40 expression in ECs and that TET1s overexpression may be the core step for OSS-induced atherosclerosis therapy.

Dosage of Dual-Protein Nutrition Differentially Impacts the Formation of Atherosclerosis in ApoE-/- Mice.

Atherosclerosis (AS) is recognized as the original cause of most cardiovascular and cerebrovascular diseases. The dual-protein (DP) nutrition that consists of soy protein and whey protein is reported to be associated with a reduction in AS; however, the relationship between DP and AS remains ambiguous. Therefore, this study aimed to verify the effect of DP on AS and explore the optimal DP intake to improve AS. ApoE-/- mice were administrated with low- (LDP), middle- (MDP), and high-dose (HDP) DP. The MDP group exhibited significant improvements in AS. In terms of lipid metabolism, the levels of plasma total triglyceride and LDL-C and the mRNA expression levels of Cyp7a1 and PCSK9 were markedly tuned in the MDP group. In addition, the MDP treatment group had a substantially lower inflammatory response and better intestinal barrier function than LDP and HDP groups. The species richness demonstrated by the Chao1 index was distinctly increased in the MDP group, and the relative abundance of intestinal-permeability-protective microbes Blautia and Akkermansia was significantly elevated. In summary, an adequate intake of DP was able to counteract atherosclerosis development in ApoE-/- mice, and this study provides a scientific theoretical basis for the application of DP in the food and pharmaceutical fields.