Human amniotic epithelial stem cell-derived dopaminergic neuron-like cells ameliorate motor dysfunction in a rat model of Parkinson's disease.

AIMS: Parkinson's disease (PD) remains a substantial clinical challenge due to the progressive loss of midbrain dopaminergic (DA) neurons in nigrostriatal pathway. In this study, human amniotic epithelial stem cells (hAESCs)-derived dopaminergic neuron-like cells (hAESCs-DNLCs) were generated, with the aim of providing new therapeutic approach to PD. MATERIALS AND METHODS: hAESCs, which were isolated from discarded placentas, were induced to differentiate into hAESCs-DNLCs by following a "two stages" induction protocol. The differentiation efficiency was assessed by quantitative real-time PCR (qRT-PCR), immunocytochemistry (ICC), and ELISA. Immunogenicity, cell viability and tumorigenicity of hAESCs-DNLC were analyzed before in vivo experiments. Subsequently, hAESCs-DNLCs were transplanted into PD rats, behavioral tests were monitored after graft, and the regeneration of DA neurons was detected by immunohistochemistry (IHC). Furthermore, to trace hAESCs-DNLCs in vivo, cells were pre-labeled with PKH67 green fluorescence. KEY FINDINGS: hAESCs were positive for pluripotent markers and highly expressed neural stem cells (NSCs) markers. Based on this, we established an induction method reliably generates hAESCs-DNLCs, which was evidenced by epithelium-to-neuron morphological changes, elevated expressions of neuronal and DA neuronal markers, and increased secretion of dopamine. Moreover, hAESCs-DNLCs maintained high cell viability, no tumorigenicity and low immunogenicity, suggesting hAESCs-DNLCs an attractive implant for PD therapy. Transplantation of hAESCs-DNLCs into PD rats significantly ameliorated motor disorders, as well as enhanced the reinnervation of TH+ DA neurons in nigrostriatal pathway. SIGNIFICANCE: Our study has demonstrated evident therapeutic effects of hAESCs-DNLCs, and provides a safe and promising solution for PD.

Human amniotic epithelial stem cell is a cell therapy candidate for preventing acute graft-versus-host disease.

Graft-versus-host disease (GVHD), an immunological disorder that arises from donor T cell activation through recognition of host alloantigens, is the major limitation in the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Traditional immunosuppressive agents can relieve GVHD, but they induce serious side effects. It is highly required to explore alternative therapeutic strategy. Human amniotic epithelial stem cells (hAESCs) were recently considered as an ideal source for cell therapy with special immune regulatory property. In this study, we evaluated the therapeutic role of hAESCs in the treatment of GVHD, based on our previous developed cGMP-grade hAESCs product. Humanized mouse model of acute GVHD (aGVHD) was established by injection of huPBMCs via the tail vein. For prevention or treatment of aGVHD, hAESCs were injected to the mice on day -1 or on day 7 post-PBMC infusion, respectively. We showed that hAESCs infusion significantly alleviated the disease phenotype, increased the survival rate of aGVHD mice, and ameliorated pathological injuries in aGVHD target organs. We demonstrated that hAESCs directly induced CD4+ T cell polarization, in which Th1 and Th17 subsets were downregulated, and Treg subset was elevated. Correspondingly, the levels of a series of pro-inflammatory cytokines were reduced while the levels of the anti-inflammatory cytokines were upregulated in the presence of hAESCs. We found that hAESCs regulated CD4+ subset polarization in a paracrine mode, in which TGFß and PGE2 were selectively secreted to mediate Treg elevation and Th1/Th17 inhibition, respectively. In addition, transplanted hAESCs preserved the graft-versus-leukemia (GVL) effect by inhibiting leukemia cell growth. More intriguingly, hAESCs infusion in HSCT patients displayed potential anti-GVHD effect with no safety concerns and confirmed the immunoregulatory mechanisms in the preclinical study. We conclude that hAESCs infusion is a promising therapeutic strategy for post-HSCT GVHD without compromising the GVL effect. The clinical trial was registered at www.clinicaltrials.gov as #NCT03764228.

SUMOylation Fine-Tunes Endothelial HEY1 in the Regulation of Angiogenesis.

BACKGROUND: Angiogenesis, which plays a critical role in embryonic development and tissue repair, is controlled by a set of angiogenic signaling pathways. As a TF (transcription factor) belonging to the basic helix-loop-helix family, HEY (hairy/enhancer of split related with YRPW motif)-1 (YRPW motif, abbreviation of 4 highly conserved amino acids in the motif) has been identified as a key player in developmental angiogenesis. However, the precise mechanisms underlying HEY1's actions in angiogenesis remain largely unknown. Our previous studies have suggested a potential role for posttranslational SUMOylation in the dynamic regulation of vascular development and organization. METHODS: Immunoprecipitation, mass spectrometry, and bioinformatics analysis were used to determine the biochemical characteristics of HEY1 SUMOylation. The promoter-binding capability of HEY1 was determined by chromatin immunoprecipitation, dual luciferase, and electrophoretic mobility shift assays. The dimerization pattern of HEY1 was determined by coimmunoprecipitation. The angiogenic capabilities of endothelial cells were assessed by CCK-8 (cell counting kit-8), 5-ethynyl-2-deoxyuridine staining, wound healing, transwell, and sprouting assays. Embryonic and postnatal vascular growth in mouse tissues, matrigel plug assay, cutaneous wound healing model, oxygen-induced retinopathy model, and tumor angiogenesis model were used to investigate the angiogenesis in vivo. RESULTS: We identified intrinsic endothelial HEY1 SUMOylation at conserved lysines by TRIM28 (tripartite motif containing 28) as the unique E3 ligase. Functionally, SUMOylation facilitated HEY1-mediated suppression of angiogenic RTK (receptor tyrosine kinase) signaling and angiogenesis in primary human endothelial cells and mice with endothelial cell-specific expression of wild-type HEY1 or a SUMOylation-deficient HEY1 mutant. Mechanistically, SUMOylation facilitates HEY1 homodimer formation, which in turn preserves HEY1's DNA-binding capability via recognition of E-box promoter elements. Therefore, SUMOylation maintains HEY1's function as a repressive TF controlling numerous angiogenic genes, including RTKs and Notch pathway components. Proangiogenic stimuli induce HEY1 deSUMOylation, leading to heterodimerization of HEY1 with HES (hairy and enhancer of split)-1, which results in ineffective DNA binding and loss of HEY1's angiogenesis-suppressive activity. CONCLUSIONS: Our findings demonstrate that reversible HEY1 SUMOylation is a molecular mechanism that coordinates endothelial angiogenic signaling and angiogenesis, both in physiological and pathological milieus, by fine-tuning the transcriptional activity of HEY1. Specifically, SUMOylation facilitates the formation of the HEY1 transcriptional complex and enhances its DNA-binding capability in endothelial cells.

Biomimetic Corneal Stroma for Scarless Corneal Wound Healing via Structural Restoration and Microenvironment Modulation.

Corneal injury-induced stromal scarring causes the most common subtype of corneal blindness, and there is an unmet need to promote scarless corneal wound healing. Herein, a biomimetic corneal stroma with immunomodulatory properties is bioengineered for scarless corneal defect repair. First, a fully defined serum-free system is established to derive stromal keratocytes (hAESC-SKs) from a current Good Manufacturing Practice (cGMP)-grade human amniotic epithelial stem cells (hAESCs), and RNA-seq is used to validate the phenotypic transition. Moreover, hAESC-SKs are shown to possess robust immunomodulatory properties in addition to the keratocyte phenotype. Inspired by the corneal stromal extracellular matrix (ECM), a photocurable gelatin-based hydrogel is fabricated to serve as a scaffold for hAESC-SKs for bioengineering of a biomimetic corneal stroma. The rabbit corneal defect model is used to confirm that this biomimetic corneal stroma rapidly restores the corneal structure, and effectively reshapes the tissue microenvironment via proteoglycan secretion to promote transparency and inhibition of the inflammatory cascade to alleviate fibrosis, which synergistically reduces scar formation by ≈75% in addition to promoting wound healing. Overall, the strategy proposed here provides a promising solution for scarless corneal defect repair.

A 3D-Printed Dual Driving Forces Scaffold with Self-Promoted Cell Absorption for Spinal Cord Injury Repair.

Stem cells play critical roles in cell therapies and tissue engineering for nerve repair. However, achieving effective delivery of high cell density remains a challenge. Here, a novel cell delivery platform termed the hyper expansion scaffold (HES) is developed to enable high cell loading. HES facilitated self-promoted and efficient cell absorption via a dual driving force model. In vitro tests revealed that the HES rapidly expanded 80-fold in size upon absorbing 2.6 million human amniotic epithelial stem cells (hAESCs) within 2 min, representing over a 400% increase in loading capacity versus controls. This enhanced uptake benefited from macroscopic swelling forces as well as microscale capillary action. In spinal cord injury (SCI) rats, HES-hAESCs promoted functional recovery and axonal projection by reducing neuroinflammation and improving the neurotrophic microenvironment surrounding the lesions. In summary, the dual driving forces model provides a new rationale for engineering hydrogel scaffolds to facilitate self-promoted cell absorption. The HES platform demonstrates great potential as a powerful and efficient vehicle for delivering high densities of hAESCs to promote clinical treatment and repair of SCI.

Endothelial FIS1 DeSUMOylation Protects Against Hypoxic Pulmonary Hypertension.

BACKGROUND: Hypoxia is a major cause and promoter of pulmonary hypertension (PH), a representative vascular remodeling disease with poor prognosis and high mortality. However, the mechanism underlying how pulmonary arterial system responds to hypoxic stress during PH remains unclear. Endothelial mitochondria are considered signaling organelles on oxygen tension. Results from previous clinical research and our studies suggested a potential role of posttranslational SUMOylation (small ubiquitin-like modifier modification) in endothelial mitochondria in hypoxia-related vasculopathy. METHODS: Chronic hypoxia mouse model and Sugen/hypoxia rat model were employed as PH animal models. Mitochondrial morphology and subcellular structure were determined by transmission electron and immunofluorescent microscopies. Mitochondrial metabolism was determined by mitochondrial oxygen consumption rate and extracellular acidification rate. SUMOylation and protein interaction were determined by immunoprecipitation. RESULTS: The involvement of SENP1 (sentrin-specific protease 1)-mediated SUMOylation in mitochondrial remodeling in the pulmonary endothelium was identified in clinical specimens of hypoxia-related PH and was verified in human pulmonary artery endothelial cells under hypoxia. Further analyses in clinical specimens, hypoxic rat and mouse PH models, and human pulmonary artery endothelial cells and human embryonic stem cell-derived endothelial cells revealed that short-term hypoxia-induced SENP1 translocation to endothelial mitochondria to regulate deSUMOylation (the reversible process of SUMOylation) of mitochondrial fission protein FIS1 (mitochondrial fission 1), which facilitated FIS1 assembling with fusion protein MFN2 (mitofusin 2) and mitochondrial gatekeeper VDAC1 (voltage-dependent anion channel 1), and the membrane tethering activity of MFN2 by enhancing its oligomerization. Consequently, FIS1 deSUMOylation maintained the mitochondrial integrity and endoplasmic reticulum-mitochondria calcium communication across mitochondrial-associated membranes, subsequently preserving pulmonary endothelial function and vascular homeostasis. In contrast, prolonged hypoxia disabled the FIS1 deSUMOylation by diminishing the availability of SENP1 in mitochondria via inducing miR (micro RNA)-138 and consequently resulted in mitochondrial dysfunction and metabolic reprogramming in pulmonary endothelium. Functionally, introduction of viral-packaged deSUMOylated FIS1 within pulmonary endothelium in mice improved pulmonary endothelial dysfunction and hypoxic PH development, while knock-in of SUMO (small ubiquitin-like modifier)-conjugated FIS1 in mice exaggerated the diseased cellular and tissue phenotypes. CONCLUSIONS: By maintaining endothelial mitochondrial homeostasis, deSUMOylation of FIS1 adaptively preserves pulmonary endothelial function against hypoxic stress and consequently protects against PH. The FIS1 deSUMOylation-SUMOylation transition in pulmonary endothelium is an intrinsic pathogenesis of hypoxic PH.

Acetate controls endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT), a process initiated by activation of endothelial TGF-ß signaling, underlies numerous chronic vascular diseases and fibrotic states. Once induced, EndMT leads to a further increase in TGF-ß signaling, thus establishing a positive-feedback loop with EndMT leading to more EndMT. Although EndMT is understood at the cellular level, the molecular basis of TGF-ß-driven EndMT induction and persistence remains largely unknown. Here, we show that metabolic modulation of the endothelium, triggered by atypical production of acetate from glucose, underlies TGF-ß-driven EndMT. Induction of EndMT suppresses the expression of the enzyme PDK4, which leads to an increase in ACSS2-dependent Ac-CoA synthesis from pyruvate-derived acetate. This increased Ac-CoA production results in acetylation of the TGF-ß receptor ALK5 and SMADs 2 and 4 leading to activation and long-term stabilization of TGF-ß signaling. Our results establish the metabolic basis of EndMT persistence and unveil novel targets, such as ACSS2, for the potential treatment of chronic vascular diseases.

Endothelial miR-196b-5p regulates angiogenesis via the hypoxia/miR-196b-5p/HMGA2/HIF1α loop.

Angiogenesis is involved in development, reproduction, wound healing, homeostasis, and other pathophysiological events. Imbalanced angiogenesis predisposes patients to various pathological processes, such as angiocardiopathy, inflammation, and tumorigenesis. MicroRNAs (miRNAs) have been found to be important in regulating cellular processing and physiological events including angiogenesis. However, the role of miRNAs that regulate angiogenesis (angiomiRs) is not fully understood. Here, we observed a downregulation of the miR-196 family in endothelial cells upon hypoxia. Functionally, miR-196b-5p inhibited the angiogenic functions of endothelial cells in vitro and suppressed angiogenesis in Matrigel plugs and skin wound healing in vivo. Mechanistically, miR-196b-5p bound onto the 3' untranslated region (UTR) of high-mobility group AT-hook 2 (HMGA2) mRNA and repressed the translation of HMGA2, which in turn represses HIF1α accumulation in endothelial cells upon hypoxia. Together, our results establish the role of endothelial miR-196b-5p as an angiomiR that negatively regulates endothelial growth in angiogenesis via the hypoxia/miR-196b-5p/HMGA2/HIF1α loop. miR-196b-5p and its regulatory loop could be an important addition to the molecular mechanisms underlying angiogenesis and may serve as potential targets for antiangiogenic therapy.

Promoting anti-tumor immunity by targeting TMUB1 to modulate PD-L1 polyubiquitination and glycosylation.

Immune checkpoint blockade therapies targeting the PD-L1/PD-1 axis have demonstrated clear clinical benefits. Improved understanding of the underlying regulatory mechanisms might contribute new insights into immunotherapy. Here, we identify transmembrane and ubiquitin-like domain-containing protein 1 (TMUB1) as a modulator of PD-L1 post-translational modifications in tumor cells. Mechanistically, TMUB1 competes with HECT, UBA and WWE domain-containing protein 1 (HUWE1), a E3 ubiquitin ligase, to interact with PD-L1 and inhibit its polyubiquitination at K281 in the endoplasmic reticulum. Moreover, TMUB1 enhances PD-L1 N-glycosylation and stability by recruiting STT3A, thereby promoting PD-L1 maturation and tumor immune evasion. TMUB1 protein levels correlate with PD-L1 expression in human tumor tissue, with high expression being associated with poor patient survival rates. A synthetic peptide engineered to compete with TMUB1 significantly promotes antitumor immunity and suppresses tumor growth in mice. These findings identify TMUB1 as a promising immunotherapeutic target.

Mechanical and Acoustic Emission Characteristics of Coal-like Rock Specimens under Static Direct Shear and Dynamic Normal Load.

In underground engineering, shear failure is a common failure type in coal-rock mass under medium and low strain-rate disturbance loads. Analyzing the shear failure mechanical properties of coal-rock mass under dynamic normal load is significant. In order to reveal the influence of disturbance load on the shear mechanical properties of coal rock, a dynamic and static load coupling electro-hydraulic servo testing machine was used to conduct the shear tests of coal-like rock materials under dynamic and constant normal load. The amplitude of dynamic load is 10 kN and the frequency is 5 Hz. The damage process of the specimens was detected by the acoustic emission (AE) detection system. The results imply that the shear failure process of coal-like rock materials under constant normal load can be divided into four stages. The normal disturbance decreased the shear strength of the specimens and increased the shear modulus of the specimens. With the increase in normal load, the influence of disturbance on the shear strength of the specimen decreased. By analyzing the AE parameters, it was found that the dynamic load made the internal damage of the specimen more severe during the shear failure process. The damage variable was calculated by AE cumulative energy, and the damage evolution was divided into three stages. The shear failure mechanism of the specimen was judged by RA (rise time/amplitude) and AF (average frequency). It was found that from the elastic deformation stage to the unstable development fracture stage, the proportion of shear fracture increased. When the dynamic normal load was 10 kN and 30 kN, the fracture was mainly shear fracture; When the dynamic normal load was 50 kN, the fracture was mainly tensile or mixed fracture. The dynamic normal load affects the shear strength and failure mechanism. Therefore, the influence of disturbance load on coal-rock mass strength cannot be ignored in underground engineering.

Study on Mechanical Properties and Weakening Mechanism of Acid Corrosion Lamprophyre.

In order to study the weakening mechanism and mechanical behaviors of hard lamprophyre of Carboniferous Permian coal-bearing strata in China's mining area, lamprophyre samples were subjected to static rock dissolution experiments with pH values of 0, 2, and 4. The acid corrosion mechanism of lamprophyre was revealed from the weight changes of samples, characteristics of solution ion concentration, and macro-mechanical properties. The experimental results show that reaction occurred between lamprophyre and acid solution. With the increasing concentration of H+, the reaction was more intense, the degree of acid etching was higher, and the weight loss was greater. The internal damage induced by acid etching results in the slow extension of the compaction stage of stress-strain curve of uniaxial compression, and the obvious deterioration of mechanical properties of the lamprophyre. The uniaxial compressive strength of the lamprophyre in the dry state is 132 MPa, which decreased to 39 MPa under the acid etching condition, showing significant mudding characteristics. Dolomite (CaMg(CO3)2 with 19.63%) and orthoclase (KAlSi3O8 with 31.4%) in lamprophyre are the major minerals constituents involved in acidification reaction. Photomicrograph recorded from SEM studies reveals that the dissolution effect was directly related to the concentration of H+ in the solution. The dissolution effect was from the surface to the inside. The small dissolution pores became larger and continuously expanded, then finally formed a skeleton structure dominated by quartz. The content of K+, Ca2+, and Mg2+ in the solution after acid etching reaction indicates that the acidified product of orthoclase is colloidal H2SiO3, which adhered to the surface of samples during acid etching and hinders the further acidification of minerals. The dissolution of dolomite and orthoclase under acidic conditions directly leads to the damage of their structure and further promotes the water-rock interaction, which is the fundamental reason for the weakening of the mechanical properties of lamprophyre.

Therapeutic Effect of Biomimetic Scaffold Loaded with Human Amniotic Epithelial Cell-Derived Neural-like Cells for Spinal Cord Injury.

Spinal cord injury (SCI) results in devastating consequences for the motor and sensory function of patients due to neuronal loss and disrupted neural circuits, confronting poor prognosis and lack of effective therapies. A new therapeutic strategy is urgently required. Here, human amniotic epithelial cells (hAEC), featured with immunocompatibility, non-tumorgenicity and no ethical issues, were induced into neural-like cells by a compound cocktail, as evidenced with morphological change and the expression of neural cell markers. Interestingly, the hAEC-neural-like cells maintain the characteristic of low immunogenicity as hAEC. Aiming at SCI treatment in vivo, we constructed a 3D-printed GelMA hydrogel biomimetic spinal cord scaffold with micro-channels, in which hAEC-neural-like cells were well-induced and grown. In a rat full transection SCI model, hAEC-neural-like cell scaffolds that were implanted in the lesion demonstrated significant therapeutic effects; the neural circuit and hindlimb locomotion were partly recovered compared to little affection in the SCI rats receiving an empty scaffold or a sham implantation operation. Thus, the establishment of hAEC-neural-like cell biomimetic scaffolds may provide a safe and effective treatment strategy for SCI.

Repair of Retinal Degeneration by Human Amniotic Epithelial Stem Cell-Derived Photoreceptor-like Cells.

The loss of photoreceptors is a major event of retinal degeneration that accounts for most cases of untreatable blindness globally. To date, there are no efficient therapeutic approaches to treat this condition. In the present study, we aimed to investigate whether human amniotic epithelial stem cells (hAESCs) could serve as a novel seed cell source of photoreceptors for therapy. Here, a two-step treatment with combined Wnt, Nodal, and BMP inhibitors, followed by another cocktail of retinoic acid, taurine, and noggin induced photoreceptor-like cell differentiation of hAESCs. The differentiated cells demonstrated the morphology and signature marker expression of native photoreceptor cells and, intriguingly, bore very low levels of major histocompatibility complex (MHC) class II molecules and a high level of non-classical MHC class I molecule HLA-G. Importantly, subretinal transplantation of the hAESCs-derived PR-like cells leads to partial restoration of visual function and retinal structure in Royal College of Surgeon (RCS) rats, the classic preclinical model of retinal degeneration. Together, our results reveal hAESCs as a potential source of functional photoreceptor cells; the hAESCs-derived photoreceptor-like cells could be a promising cell-replacement candidate for therapy of retinal degeneration diseases.

Tacrolimus Causes Hypertension by Increasing Vascular Contractility via RhoA (Ras Homolog Family Member A)/ROCK (Rho-Associated Protein Kinase) Pathway in Mice.

BACKGROUND: To provide tacrolimus is first-line treatment after liver and kidney transplantation. However, hypertension and nephrotoxicity are common tacrolimus side effects that limit its use. Although tacrolimus-related hypertension is well known, the underlying mechanisms are not. Here, we test whether tacrolimus-induced hypertension involves the RhoA (Ras homolog family member A)/ROCK (Rho-associated protein kinase) pathway in male C57Bl/6 mice. METHODS: Intra-arterial blood pressure was measured under anesthesia. The reactivity of renal afferent arterioles and mesenteric arteries were assessed in vitro using microperfusion and wire myography, respectively. RESULTS: Tacrolimus induced a transient rise in systolic arterial pressure that was blocked by the RhoA/ROCK inhibitor Fasudil (12.0±0.9 versus 3.2±0.7; P<0.001). Moreover, tacrolimus reduced the glomerular filtration rate, which was also prevented by Fasudil (187±20 versus 281±8.5; P<0.001). Interestingly, tacrolimus enhanced the sensitivity of afferent arterioles and mesenteric arteries to Ang II (angiotensin II), likely due to increased intracellular Ca2+ mobilization and sensitization. Fasudil prevented increased Ang II-sensitivity and blocked Ca2+ mobilization and sensitization. Preincubation of mouse aortic vascular smooth muscle cells with tacrolimus activated the RhoA/ROCK/MYPT-1 (myosin phosphatase targeting subunit 1) pathway. Further, tacrolimus increased cytoplasmic reactive oxygen species generation in afferent arterioles (107±5.9 versus 163±6.4; P<0.001) and in cultured mouse aortic vascular smooth muscle cells (100±7.5 versus 160±23.2; P<0.01). Finally, the reactive oxygen species scavenger Tempol inhibited tacrolimus-induced Ang II hypersensitivity in afferent arterioles and mesenteric arteries. CONCLUSIONS: The RhoA/ROCK pathway may play an important role in tacrolimus-induced hypertension by enhancing Ang II-specific vasoconstriction, and reactive oxygen species may participate in this process by activating the RhoA/ROCK pathway.

FGFR1 SUMOylation coordinates endothelial angiogenic signaling in angiogenesis.

Angiogenesis contributes fundamentally to embryonic development, tissue homeostasis, and wound healing. Basic fibroblast growth factor (FGF2) is recognized as the first proangiogenic molecule discovered, and it facilitates angiogenesis by activating FGF receptor 1 (FGFR1) signaling in endothelial cells. However, the precise roles of FGFR and the FGF/FGFR signaling axis in angiogenesis remain unclear, especially because of the contradictory phenotypes of in vivo FGF and FGFR gene deficiency models. Our previous study results suggested a potential role of posttranslational small ubiquitin-like modifier modification (SUMOylation), with highly dynamic regulatory features, in vascular development and disorder. Here, we identified SENP1-regulated endothelial FGFR1 SUMOylation at conserved lysines responding to proangiogenic stimuli, while SENP1 functioned as the deSUMOylase. Hypoxia-enhanced FGFR1 SUMOylation restricted the tyrosine kinase activation of FGFR1 by modulating the dimerization of FGFR1 and FGFR1 binding with its phosphatase PTPRG. Consequently, it facilitated the recruitment of FRS2α to VEGFR2 but limited additional recruitment of FRS2α to FGFR1, supporting the activation of VEGFA/VEGFR2 signaling in endothelial cells. Furthermore, SUMOylation-defective mutation of FGFR1 resulted in exaggerated FGF2/FGFR1 signaling but suppressed VEGFA/VEGFR2 signaling and the angiogenic capabilities of endothelial cells, which were rescued by FRS2α overexpression. Reduced angiogenesis and endothelial sprouting in mice bearing an endothelial-specific, FGFR1 SUMOylation-defective mutant confirmed the functional significance of endothelial FGFR1 SUMOylation in vivo. Our findings identify the reversible SUMOylation of FGFR1 as an intrinsic fine-tuned mechanism in coordinating endothelial angiogenic signaling during neovascularization; SENP1-regulated FGFR1 SUMOylation and deSUMOylation controls the competitive recruitment of FRS2α by FGFR1 and VEGFR2 to switch receptor-complex formation responding to hypoxia and normoxia angiogenic environments.

ALX1-transcribed LncRNA AC132217.4 promotes osteogenesis and bone healing via IGF-AKT signaling in mesenchymal stem cells.

The osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) is critical for bone formation and regeneration. A high non-/delayed-union rate of fracture healing still occurs in specific populations, implying an urgent need to discover novel targets for promoting osteogenesis and bone regeneration. Long non-coding (lnc)RNAs are emerging regulators of multiple physiological processes, including osteogenesis. Based on differential expression analysis of RNA sequencing data, we found that lncRNA AC132217.4, a 3'UTR-overlapping lncRNA of insulin growth factor 2 (IGF2), was highly induced during osteogenic differentiation of BMSCs. Afterward, both gain-of-function and loss-of-function experiments proved that AC132217.4 promotes osteoblast development from BMSCs. As for its molecular mechanism, we found that AC132217.4 binds with IGF2 mRNA to regulate its expression and downstream AKT activation to control osteoblast maturation and function. Furthermore, we identified two splicing factors, splicing component 35 KDa (SC35) and heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), which regulate the biogenesis of AC132217.4 at the post-transcriptional level. We also identified a transcription factor, ALX1, which regulates AC132217.7 expression at the transcriptional level to promote osteogenesis. Importantly, in-vivo over-expression of AC132217.4 essentially promotes the bone healing process in a murine tibial drill-hole model. Our study demonstrates that lncRNA AC132217.4 is a novel anabolic regulator of BMSC osteogenesis and could be a plausible therapeutic target for improving bone regeneration.

Human Amniotic Epithelial Stem Cell-Derived Retinal Pigment Epithelium Cells Repair Retinal Degeneration.

Age-related macular degeneration (AMD), featured with dysfunction and loss of retinal pigment epithelium (RPE), is lacking efficient therapeutic approaches. According to our previous studies, human amniotic epithelial stem cells (hAESCs) may serve as a potential seed cell source of RPE cells for therapy because they have no ethical concerns, no tumorigenicity, and little immunogenicity. Herein, trichostatin A and nicotinamide can direct hAESCs differentiation into RPE like cells. The differentiated cells display the morphology, marker expression and cellular function of the native RPE cells, and noticeably express little MHC class II antigens and high level of HLA-G. Moreover, visual function and retinal structure of Royal College of Surgeon (RCS) rats, a classical animal model of retinal degeneration, were rescued after subretinal transplantation with the hAESCs-derived RPE like cells. Our study possibly makes some contribution to the resource of functional RPE cells for cell therapy. Subretinal transplantation of hAESCs-RPE could be an optional therapeutic strategy for retinal degeneration diseases.

Programming living sensors for environment, health and biomanufacturing.

Synthetic biology offers new tools and capabilities of engineering cells with desired functions for example as new biosensing platforms leveraging engineered microbes. In the last two decades, bacterial cells have been programmed to sense and respond to various input cues for versatile purposes including environmental monitoring, disease diagnosis and adaptive biomanufacturing. Despite demonstrated proof-of-concept success in the laboratory, the real-world applications of microbial sensors have been restricted due to certain technical and societal limitations. Yet, most limitations can be addressed by new technological developments in synthetic biology such as circuit design, biocontainment and machine learning. Here, we summarize the latest advances in synthetic biology and discuss how they could accelerate the development, enhance the performance and address the present limitations of microbial sensors to facilitate their use in the field. We view that programmable living sensors are promising sensing platforms to achieve sustainable, affordable and easy-to-use on-site detection in diverse settings.

Synthetic protein-binding DNA sponge as a tool to tune gene expression and mitigate protein toxicity.

Versatile tools for gene expression regulation are vital for engineering gene networks of increasing scales and complexity with bespoke responses. Here, we investigate and repurpose a ubiquitous, indirect gene regulation mechanism from nature, which uses decoy protein-binding DNA sites, named DNA sponge, to modulate target gene expression in Escherichia coli. We show that synthetic DNA sponges can be designed to reshape the response profiles of gene circuits, lending multifaceted tuning capacities including reducing basal leakage by >20-fold, increasing system output amplitude by >130-fold and dynamic range by >70-fold, and mitigating host growth inhibition by >20%. Further, multi-layer DNA sponges for decoying multiple regulatory proteins provide an additive tuning effect on the responses of layered circuits compared to single-layer sponges. Our work shows synthetic DNA sponges offer a simple yet generalizable route to systematically engineer the performance of synthetic gene circuits, expanding the current toolkit for gene regulation with broad potential applications.