Higher matrix stiffness promotes VSMC senescence by affecting mitochondria-ER contact sites and mitochondria/ER dysfunction.

Abdominal aortic aneurysm (AAA) is a prevalent condition characterized by the weakening and bulging of the abdominal aorta. This study aimed to investigate the impact of a stiff matrix on vascular smooth muscle cells (VSMCs) in AAA development. Bioinformatics analysis revealed that differentially expressed genes (DEGs) in VSMCs of an AAA mouse model were enriched in cellular senescence and related pathways. To simulate aging-related changes, VSMCs were cultured on stiff matrices, and compared to those on soft matrices, the VSMCs cultured on stiff matrices exhibited cellular senescence. Furthermore, the mutual distance between mitochondria and endoplasmic reticulum (ER) in VSMCs was increased, indicating altered mitochondria-endoplasmic reticulum contacts (MERCs). The observed upregulation of reactive oxygen species (ROS) levels, antioxidant gene expression, and decreased mitochondrial membrane potential suggested the presence of mitochondrial dysfunction in VSMCs cultured on a stiff matrix. Additionally, the induction of ER stress-related genes indicated ER dysfunction. These findings collectively indicated impaired functionality of both mitochondria and ER in VSMCs cultured on a stiff matrix. Moreover, our data revealed that high lipid levels exacerbated the effects of high matrix stiffness on VSMCs senescence, MERC sites, and mitochondria/ER dysfunction. Importantly, treatment with the antilipemic agent CI-981 effectively reversed these detrimental effects. These findings provide insights into the role of matrix stiffness, mitochondrial dysfunction, ER stress, and lipid metabolism in AAA development, suggesting potential therapeutic targets for intervention.

Single Impact Injury of Vertebral Endplates Without Structural Disruption, Initiates Disc Degeneration Through Piezo1 Mediated Inflammation and Metabolism Dysfunction.

STUDY DESIGN: In vitro experimental study. OBJECTIVE: To establish an axial impact injury model of intervertebral disc (IVD) and to investigate if a single impact injury without endplate structural disruption could initiate intervertebral disc degeneration (IDD), and what is the roles of Piezo1 in this process. SUMMARY OF BACKGROUND DATA: Although IDD process has been confirmed to be associated with structural failures such as endplate fractures, whether a single impact injury of the endplates without structural disruption could initiate IDD remains controversial. Previous studies reported that Piezo1 mediated inflammation participated in the progression of IDD induced by mechanical stretch; however, the roles of Piezo1 in IVD impact injury remain unknown. METHODS: Rats spinal segments were randomly assigned into Control, Low, and High Impact groups, which were subjected to pure axial impact loading using a custom-made apparatus, and cultured for 14 days. The degenerative process was investigated by using histomorphology, real-time Polymerase Chain Reaction(PCR), western-blot, immunofluorescence, and energy metabolism of IVD cell. The effects of Piezo1 were investigated by using siRNA transfection, real-time PCR, western-blot, and immunofluorescence. RESULTS: The discs in both of the impact groups presented degenerative changes after 14 days, which showed significant up-regulation of Piezo1, NLRP3 inflammasome, the catabolic (MMP-9, MMP-13), and pro-inflammatory gene (IL-1ß) expression than that of the control group (P < 0.05), accompanied by significantly increased release of ATP, lactate, nitric oxide (NO), and glucose consumption of IVD cells at first 7 days. Silencing Piezo1 reduced the activation of NLRP3 inflammasome and IL-1ß expression in the nucleus pulposus induced by impact injury. CONCLUSION: It demonstrated that not only fracture of the endplate but also a single impact injury without structural impairment could also initiate IDD, which might be mediated by activation of Piezo1 induced inflammation and abnormal energy metabolism of IVD cells.Level of Evidence: N/A.

Spontaneous fundus lesions in elderly monkeys: An ideal model for age-related macular degeneration and high myopia clinical research.

AIMS: Age-related macular degeneration (AMD) and high myopia are frequent causes of progressive visual impairment, so it is critical to identify animal models with resembling human retinal physiology, AMD and high myopia pathological features for therapeutic studies. MAIN METHODS: We screened elderly cynomolgus monkeys for fundus lesions by slit-lamp biomicroscope combined with fundus pre-set lens and further examined positive cases by color fundus photography (CFP), optical coherence tomography (OCT), fundus fluorescein angiography (FFA), streak retinoscopy, and A-scan ultrasonography. KEY FINDINGS: Among the 156 animals examined, 10 males and 5 females (30 eyes) exhibited fundus abnormalities (9.6% prevalence). Multi-modal imaging revealed drusen in 20 eyes of 11 animals (prevalence rate of 7.1%), tessellated fundus in 22 eyes of 11 animals, and myopia choroidal neovascularization (CNV) in 4 eyes of 3 animals. SIGNIFICANCE: Aged cynomolgus monkeys exhibit spontaneous fundus lesions resembling human AMD and high myopia, which could be an ideal model for clinical research.

Migration of pre-induced human peripheral blood mononuclear cells from the transplanted to contralateral eye in mice.

BACKGROUND: Retina diseases may lead to blindness as they often afflict both eyes. Stem cell transplantation into the affected eye(s) is a promising therapeutic strategy for certain retinal diseases. Human peripheral blood mononuclear cells (hPBMCs) are a good source of stem cells, but it is unclear whether pre-induced hPBMCs can migrate from the injected eye to the contralateral eye for bilateral treatment. We examine the possibility of bilateral cell transplantation from unilateral cell injection. METHODS: One hundred and sixty-one 3-month-old retinal degeneration 1 (rd1) mice were divided randomly into 3 groups: an untreated group (n = 45), a control group receiving serum-free Dulbecco's modified Eagle's medium (DMEM) injection into the right subretina (n = 45), and a treatment group receiving injection of pre-induced hPBMCs into the right subretina (n = 71). Both eyes were examined by full-field electroretinogram (ERG), immunofluorescence, flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) at 1 and 3 months post-injection. RESULTS: At both 1 and 3 months post-injection, labeled pre-induced hPBMCs were observed in the retinal inner nuclear layer of the contralateral (left untreated) eye as well as the treated eye as evidenced by immunofluorescence staining for a human antigen. Flow cytometry of fluorescently label cells and qRT-PCR of hPBMCs genes confirmed that transplanted hPBMCs migrated from the treated to the contralateral untreated eye and remained viable for up to 3 months. Further, full-field ERG showed clear light-evoked a and b waves in both treated and untreated eyes at 3 months post-transplantation. Labeled pre-induced hPBMCs were also observed in the contralateral optic nerve but not in the blood circulation, suggesting migration via the optic chiasm. CONCLUSION: It may be possible to treat binocular eye diseases by unilateral stem cell injection.

Single-Cell RNA-Seq Analysis Reveals Macrophage Involved in the Progression of Human Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) has been considered as the primary pathological mechanism that underlies low back pain. Understanding the molecular mechanisms underlying human IDD is imperative for making strategies to treat IDD-related diseases. Herein, we report the molecular programs, lineage progression patterns, and paths of cellular communications during the progression of IDD using single-cell RNA sequencing (scRNA-seq) on nucleus pulposus (NP) cells from patients with different grades of IDD undergoing discectomy. New subtypes of cells and cell-type-specific gene signatures of the metabolic homeostatic NP cells (Met NPC), adhesive NP cells (Adh NPC), inflammatory response NP cells (IR NPC), endoplasmic reticulum stress NP cells (ERS NPC), fibrocartilaginous NP cells (Fc NPC), and CD70 and CD82+ progenitor NP cells (Pro NPC) were identified. In the late stage of IDD, the IR NPC and Fc NPC account for a large proportion of NPC. Importantly, immune cells including macrophages, T cells, myeloid progenitors, and neutrophils were also identified, and further analysis showed that significant intercellular interaction between macrophages and Pro NPC occurred via MIF (macrophage migration inhibitory factor) and NF-kB signaling pathways during the progression of IDD. In addition, dynamic polarization of macrophage M1 and M2 cell subtypes was found in the progression of IDD, and gene set functional enrichment analysis suggested a significant role of the macrophage polarization in regulating cell metabolism, especially the Pro NPC. Finally, we found that the NP cells in the late degenerative stage were mainly composed of the cell types related to inflammatory and endoplasmic reticulum (ER) response, and fibrocartilaginous activity. Our results provided new insights into the identification of NP cell populations at single-cell resolution and at the relatively whole-transcriptome scale, accompanied by cellular communications between immune cells and NP cells, and discriminative markers in relation to specific cell subsets. These new findings present clues for effective and functional manipulation of human IDD-related bioremediation and healthcare.

Cationic Polymer Brush-Modified Carbon Nanotube-Meditated eRNA LINC02569 Silencing Attenuates Nucleus Pulposus Degeneration by Blocking NF-κB Signaling Pathway and Alleviate Cell Senescence.

Enhancer RNAs (eRNAs) are noncoding RNAs that synthesized at active enhancers. eRNAs have important regulatory characteristics and appear to be significant for maintenance of cell identity and information processing. Series of functional eRNAs have been identified as potential therapeutic targets for multiple diseases. Nevertheless, the role of eRNAs on intervertebral disc degeneration (IDD) is still unknown yet. Herein, we utilized the nucleus pulposus samples of patients and identified a key eRNA (LINC02569) with the Arraystar eRNA Microarray. LINC02569 mostly locates in nucleus and plays an important role in the progress of IDD by activating nuclear factor kappa-B (NF-κB) signaling pathway. We used a cationic polymer brush coated carbon nanotube (oCNT-pb)-based siRNA delivery platform that we previously designed, to transport LINC02569 siRNA (si-02569) to nucleus pulposus cells. The siRNA loaded oCNT-pb accumulated in nucleus pulposus cells with lower toxicity and higher transfection efficiency, compared with the traditional siRNA delivery system. Moreover, the results showed that the delivery of si-02569 significantly alleviated the inflammatory response in the nucleus pulposus cells via inhibiting P65 phosphorylation and preventing its transfer into the nucleus, and meanwhile alleviated cell senescence by decreasing the expression of P21. Altogether, our results highlight that eRNA (LINC02569) plays important role in the progression of IDD and could be a potential therapeutic target for alleviation of IDD.

Corneal Recovery Following Rabbit Peripheral Blood Mononuclear Cell-Amniotic Membrane Transplantation with Antivascular Endothelial Growth Factor in Limbal Stem Cell Deficiency Rabbits.

Background: Limbal stem cell deficiency (LSCD) is a refractory ocular surface disorder characterized by progressive corneal epithelial degeneration, conjunctivalization, and neovascularization, potentially leading to blindness. There are currently no effective therapeutic options for patients experiencing routine symptomatic treatment failure. Transplantation of amniotic membrane (AM) with adherent stem cells (but not bare AM transplantation alone) has shown promise in preclinical studies for ocular surface restoration. A major limitation, however, is finding a reliable stem cell source. Stem cells can be isolated from the peripheral blood mononuclear cell (PBMC) population, and these PBMC-derived stem cells have numerous advantages over allogeneic and other autologous stem cell types for therapeutic application, including relative ease of acquisition, nonimmunogenicity, and the absence of ethical issues associated with embryonic stem cells. Experiment: We examined the efficacy of autologous PBMC-AM sheet cultures combined with postoperative antiangiogenesis treatment for corneal restoration in LSCD model rabbits. Rabbit PBMCs (rPBMCs) were isolated, labeled with EdU for in vivo tracing, and then cultured on AMs in conditioned medium before transplantation. Rabbits were transplanted with bare AMs (group 1), rPBMC-AM sheets (group 2), or rPBMC-AM sheets plus postoperative treatment with the vascular endothelial growth factor antagonist bevacizumab (group 3). Corneal opacity and neovascularization were monitored by slit-lamp imaging for 8 weeks and corneas were examined histologically at 1 and 2 months. Results: Corneal opacity decreased in all three groups over 8 weeks, but was significantly lower in group 2 and even lower in group 3. Corneal neovascularization was significantly higher in group 1 throughout the observation period, and significantly lower in group 3 than group 1 and 2 by 8 weeks post-transplant. At 4 weeks, the corneal surface completed epithelialization (although thinner than normal) in group 3 but still patchy in groups 1 and 2. By 8 weeks, the epithelium in group 3 was complete and smooth, resembling a normal epithelium. Integrin ß1 as a progenitor marker was also generally higher in groups 2 and 3. Conclusions: Autologous rPBMC-AM sheets with post-transplant topical bevacizumab can effectively facilitate corneal epithelium recovery in a LSCD model, suggesting clinical utility for LSCD-related ocular surface diseases. Impact statement Limbal stem cell deficiency (LSCD) increases corneal opacity and vascularization, resulting in severe visual impairment or even blindness. Traditional surgical limbal transplant is currently the main treatment option for LSCD, but carries the risks of rejection and immunosuppressant side effects. Autologous stem cell-based therapy is a promising alternative approach, but a reliable stem cell source is a major limitation. We report that transplantation of autologous rabbit peripheral blood mononuclear cell-amniotic membrane sheets plus antivascular endothelial growth factor restored avascular transparent cornea in a rabbit LSCD model. These results demonstrate a potentially effective approach for ocular surface reconstruction in bilateral LSCD.

A novel lncRNA-miRNA-mRNA competitive endogenous RNA network for uveal melanoma prognosis constructed by weighted gene co-expression network analysis.

AIMS: Uveal melanoma (UM) is the most common and aggressive intraocular tumor in adults, and long-term survival of UM patients remains poor. Abnormal competitive endogenous RNA (ceRNA) networks promote the initiation and progression of many tumors and may thus serve as useful prognostic indicators. Here, we do a comprehensive analysis of long non-coding RNA (lncRNA)-microRNA (miRNA)-mRNA ceRNA networks as prognostic markers for UM. MATERIALS AND METHODS: The Cancer Genome Atlas UM dataset was used to identify survival-related mRNA and lncRNA modules through weighted gene co-expression network analysis (WGCNA). Prognostic miRNAs were identified using univariate Cox proportional hazard regression. We then used Cox and least absolute shrinkage and selection operator regression to screen for prognostic hub mRNAs and establish a hub ceRNA network. A nomogram of five hub mRNAs was constructed and Kaplan-Meier survival analysis performed. KEY FINDINGS: Six mRNA modules were constructed, two of which involved 1490 mRNAs that significantly correlated with survival. Among the three lncRNA modules constructed, one involved 199 survival-related lncRNAs. Five hub prognostic mRNAs were identified and a hub ceRNA network constructed, consisting of six lncRNAs, four miRNAs, and five mRNAs, with high prognostic value. SIGNIFICANCE: We describe a hub ceRNA network of survival-associated lncRNAs, miRNAs, and mRNA that may underlie a critical post-translational regulatory mechanism determining UM aggression. These hub RNAs may be valuable prognostic markers and therapeutic targets in UM.