Experimental Biology and Medicine

Aortic aneurysms are characterized by vascular inflammation, neovascularization, and extracellular matrix destruction of the aortic wall. Although experimental studies indicate a potential role of CD248 in microvessel remodeling, the functions of CD248 in human vascular pathologies remain unexplored. Here we aimed to study how CD248 interferes with pathological vascular remodeling of human aortic aneurysms. Immunofluorescent staining showed that CD248 expression was mainly localized in the CD8+ T cells infiltrating in the adventitia and media of aortic walls of patients with ascending thoracic aortic aneurysms. qPCR and immunofluorescent staining analyses revealed increased aortic CD248 expression and infiltrating CD248+CD8+ T cells in aortic aneurysms than in nonaneurysmal aortas. Flow cytometry analysis of human peripheral blood further identified a fraction of circulating CD248+ cells which was confined in the CD8+ T-cell compartment. The increased infiltrating of CD248+CD8+ T cells was coincident with reduced circulating CD248+CD8+ T cells in patients with ascending TAA when compared with patients with coronary artery diseases and healthy donors. The CD248+CD8+ T cells were characterized by upregulated IL-10 and downregulated IL-1β/INF-γ expression when compared with CD248-CD8+ T cells. Moreover, when co-cultured with human aortic endothelial cells, the CD248+CD8+ T cells not only downregulated endothelial expression of ICAM1/VCAM1 and MMP2/3 but also suppressed endothelial migration. This study shows that CD248 reduces pathological vascular remodeling via anti-inflammatory CD248+CD8+ T cells, revealing a CD248-mediated cellular mechanism against human aortic aneurysms.

Treatment of congenital pseudarthrosis of the tibia (CPT) still is full of challenges in pediatric orthopedist. Serum-derived exosomes (SDEs) have been proven to be participated in bone remodeling. However, the molecular changes in SDEs of CPT children and their pathologies have not been elucidated. In this study, SDEs were isolated and purified from CPT patients (CPT-SDEs) associated with neurofibromatosis type 1 (NF1) and normal children (Norm-SDEs). Then we obtained the proteomics profile of SDEs by combining liquid chromatography-tandem mass spectrometry (LC-MS/MS) and tandem mass tag label-based quantitation. In vitro, the efficacy of SDEs on osteoblastic differentiation of MC3T3-E1 cells and osteoclastogenesis ability of RAW264.7 cells were evaluated by quantitative real-time PCR (qRT-PCR) and cytochemical staining. In vivo, we used micro-CT to assess cortical bone mass and trabecular microstructures to reflect the influence of SDEs on bone remodeling after injection into the tail vein of rats. Based on proteomics analysis, 410 differentially expressed proteins, including 289 downregulated proteins and 121 upregulated proteins, were identified in the CPT-SDEs. These proteins have multiple biological functions associated with cellular metabolic processes, catalytic activity, and protein binding, which are important for cell differentiation and proliferation. In vitro, CPT-SDEs decreased the osteogenic differentiation of MC3T3-E1 cells and promoted the osteoclastogenesis of RAW264.7 cells. Injection of CPT-SDEs into the tail vein for two months resulted in bone loss in rats, as indicated by the decrease in trabecular and cortical bone mass. Our findings demonstrated the differences in proteins in SDEs between normal and CPT children with NF1. These differentially expressed proteins in CPT-SDEs contributed to deteriorating trabecular bone microstructures by inhibiting bone formation and stimulating bone resorption.

The long non-coding RNA colon cancer-associated transcript 1 (CCAT1) has been investigated to involve in the progression of non-small cell lung cancer (NSCLC). Thus, this study aims to explore the detailed molecular mechanisms of CCAT1 in NSCLC. The expression of CCAT1, miR-216a-5p, RAP2B, Bax, Bcl-2, and cleaved caspase 3 was detected by qRT-PCR or Western blot. Cell proliferation, apoptosis, migration, and invasion were analyzed using cell counting kit-8, flow cytometry or Transwell assays, respectively. The interaction between miR-216a-5p and CCAT1 or RAP2B was analyzed by luciferase reporter, RNA immunoprecipitation, and pull-down assays. The expression of CCAT1 was elevated in NSCLC, and CCAT1 deletion could inhibit NSCLC cell proliferation, migration, and invasion but induce apoptosis in vitro as well as imped tumor growth in vivo. MiR-216a-5p was confirmed to be a target of CCAT1, and silencing miR-216a-5p could reverse CCAT1 depletion-mediated inhibitory effects on cell tumorigenesis in NSCLC. Besides that, miR-216a-5p was decreased in NSCLC, and miR-216a-5p restoration inhibited cell tumorigenesis by regulating RAP2B, which was verified to be a target of miR-216a-5p. Additionally, co-expression analysis suggested that CCAT1 indirectly regulated RAP2B level by targeting miR-216a-5p in NSCLC cells. Taken together, CCAT1 deletion could inhibit cell progression in NSCLC through miR-216a-5p/RAP2B axis, indicating a novel pathway underlying NSCLC cell progression and providing new potential targets for NSCLC treatment.

Asymmetric dimethylarginine is an endogenous competitive inhibitor of nitric oxide synthase and marker of endothelial dysfunction, but the question remains as to whether asymmetric dimethylarginine is a marker of cardiovascular episodes or their independent risk factor. ADMA/DDAH (dimethylaminohydrolase) pathway regulates vascular endothelial growth factor (VEGF)-mediated angiogenesis due to its impact on the NO formation. The aim of the study was to assess the concentrations of asymmetric dimethylarginine and the angiogenic potential in the blood of subjects with type 2 diabetes (T2DM, n = 33) and patients with prediabetes (n = 32)—impaired fasting glycemia and/or impaired glucose tolerance (WHO criteria). The study found that both the prediabetes group and subjects with T2DM had significantly elevated concentrations of asymmetric dimethylarginine, significantly high levels of VEGF-A, low ratio of sVEGF-R1/VEGF-A, and sVEGF-R2/VEGF-A. This may suggest endothelial damage at early stages of carbohydrate metabolism dysfunction—before T2DM is diagnosed. Higher proangiogenic potential in prediabetes and T2DM patients than in healthy subjects, is not only the effect of an increase in VEGF-A levels, but also reduced inhibition of circulating receptors.

Obesity is one of major risk factors increasing chronic diseases including type II diabetes, cardiovascular diseases, and hypertension. The effects of epigallocatechin gallate (EGCG), the major active compound in green tea, on reduced obesity and improved metabolic profiles are still controversial. Furthermore, the effects of EGCG on human adipocyte lipolysis and browning of white adipocytes have not been elucidated. This study aimed to investigate the effects of EGCG on obesity, lipolysis, and browning of human white adipocytes. The results showed that, when compared to the baseline values, EGCG significantly decreased fasting plasma triglyceride levels (P < 0.05), systolic blood pressure (P < 0.05), diastolic blood pressure (P < 0.05), and serum kisspeptin levels (P < 0.05) after 8 weeks of supplement. On the other hand, supplement of EGCG in obese human subjects for 4 or 8 weeks did not decrease body weight, body mass index, waist and hip circumferences, nor total body fat mass or percentage when compared to their baseline values. The study in human adipocytes showed that EGCG did not increase the glycerol release when compared to vehicle, suggesting that it had no lipolytic effect. Furthermore, treatment of EGCG did not enhance uncoupling protein 1 (UCP1) mRNA expression in human white adipocytes when compared with treatment of pioglitazone, the peroxisome proliferator-activated receptor γ (PPAR-γ) agonist, suggesting that EGCG did not augment the browning effect of PPAR-γ on white adipocytes. This study revealed that EGCG reduced 2 metabolic risk factors which are triglyceride and blood pressure in the human experiment. We also showed a novel evidence that EGCG decreased kisspeptin levels. However, EGCG had no effects on obesity reduction in humans, lipolysis, nor browning of human white adipocytes.

Long noncoding RNAs play an important role in the occurrence, invasion, as well as metastasis of various human cancers, including hepatocellular carcinoma. Long noncoding RNAs can affect the biological functions of hepatocellular carcinoma cells by regulating various genes; however, only a small fraction of molecular mechanisms of long noncoding RNAs have been elucidated. In the present study, lnc AC010973.1 (lnc-ATG9B-4) was first identified by microarray analysis from 8 patients with hepatocellular carcinoma and confirmed by quantitative PCR in 176 patients with hepatocellular carcinoma. We demonstrated that lnc-ATG9B-4 was tightly relative to the tumorous size, TNM stages, portal vein tumor thrombus (PVTT), the tumor capsule, metastasis, degree of differentiation, and poor prognosis of hepatocellular carcinoma according to long-term follow-up data. In hepatocellular carcinoma cells, overexpression of lnc-ATG9B-4 promoted proliferation, invasion, as well as migration, while inhibiting lnc-ATG9B-4 by siRNA significantly attenuated the proliferation, invasion, as well as migration. Interestingly, lnc-ATG9B-4 increased the expression of cyclin-dependent kinase 5 (CDK5), which was closely related to the development and chemotherapy sensitivity of hepatocellular carcinoma. In summary, our results revealed that lnc-ATG9B-4 suggests an unfavorable prognosis of hepatocellular carcinoma and facilitates the proliferation, invasion, as well as migration of hepatocellular carcinoma cells by upregulating CDK5. This research suggests that lnc-ATG9B-4 may be a new biomarker for predicting the prognosis of hepatocellular carcinoma; meanwhile, targeting lnc-ATG9B-4 might serve as a potential strategy for the treatment hepatocellular carcinoma.

Temozolomide (TMZ) is the major chemotherapy agent in glioma, and isocitrate dehydrogenase (IDH) is a well-known prognostic marker in glioma. O6-methylguanine-DNA methyltransferase promoter methylation (MGMTmethyl) is a predictive biomarker in overall gliomas rather than in IDH mutant gliomas. To discover effective biomarkers that could predict TMZ efficacy in IDH mutant low-grade gliomas (LGGs), we retrieved data of IDH mutant LGGs from TMZ arm of the EORTC22033-26033 trial as the training-set (n = 83), analyzed correlations between long non-coding RNAs (lncRNAs) and progression-free survival (PFS) using Lasso-Cox regression, and created a risk score (RS) to stratify patients. We identified a three-lncRNA signature in TMZ-treated IDH mutant LGGs. All of the three lncRNAs, as well as the RS derived, were significantly correlated with PFS. Patients were classified into high-risk and low-risk groups according to RS. PFS of the high-risk group was significantly worse than that of the low-risk group (P < 0.001). AUCs of the three-, four-, and five-year survival probability predicted by RS were 0.73, 0.79, and 0.76, respectively. The predictive role of the three-lncRNA signature was further validated in an independent testing-set, the TCGA-LGGs, which resulted in a significantly worse PFS (P < 0.001) in the high-risk group. Three-, four-, and five-year survival probabilities predicted by RS were 0.65, 0.69, and 0.84, respectively. Functions of these three lncRNAs involve cell proliferation and differentiation, predicted by their targeting cancer genes. Conclusively, we created a scoring model based on the expression of three lncRNAs, which can effectively predict the survival of IDH mutant LGGs treated with TMZ.

Physiologically, the human and murine hearing systems are very similar, justifying the extensive use of mice in experimental models for hearing impairment (HI). About 340 murine HI genes have been reported; however, whether variants in all human-mouse ortholog genes contribute to HI has been rarely investigated. In humans, nearly 120 HI genes have been identified to date, with GJB2 and GJB6 variants accounting for half of congenital HI cases, of genetic origin, in populations of European and Asian ancestries, but not in most African populations. The contribution of variants in other known genes of HI among the populations of African ancestry is poorly studied and displays the lowest pick-up rate. We used whole exome sequencing (WES) to investigate pathogenic and likely pathogenic (PLP) variants in 34 novel human-mouse orthologs HI genes, in 40 individuals from Cameroon and South Africa diagnosed with non-syndromic hearing impairment (NSHI), and compared the data to WES data of 129 ethnically matched controls. In addition, protein modeling for selected PLP gene variants, gene enrichment, and network analyses were performed. A total of 4/38 murine genes, d6wsu163e, zfp719, grp152 and minar2, had no human orthologs. WES identified three rare PLP variants in 3/34 human-mouse orthologs genes in three unrelated Cameroonian patients, namely: OCM2, c.227G>C p.(Arg76Thr) and LRGI1, c.1657G>A p.(Gly533Arg) in a heterozygous state, and a PLP variant MCPH1, c.2311C>G p.(Pro771Ala) in a homozygous state. In silico functional analyses suggest that these human-mouse ortholog genes functionally co-expressed interactions with well-established HI genes: GJB2 and GJB6. The study found one homozygous variant in MCPH1, likely to explain HI in one patient, and suggests that human-mouse ortholog variants could contribute to the understanding of the physiology of hearing in humans.

Autophagy plays a crucial role in cellular development and differentiation as well as in the maintenance of homeostasis in healthy cells. Autophagy is well documented in neurodegenerative disorders, aging, and infectious diseases. However, recognizing its significance in cancer has always been challenging due to its tumor-promoting and suppressive attributes. Various modulators targeting key components of autophagy machinery directly or indirectly have been developed over the years, and have shown promising results in preclinical models. Some of these compounds are even being tested in clinical trials for safety and efficacy. A detailed review of strategies used to target autophagy in cancer is presented including our opinion on developing better therapies and outstanding issues.

Cardiovascular diseases are the leading cause of mortality and morbidity worldwide. Histone deacetylases (HDACs) play an important role in the epigenetic regulation of genetic transcription in response to stress or pathological conditions. HDACs interact with a complex co-regulatory network of transcriptional regulators, deacetylate histones or non-histone proteins, and modulate gene expression in the heart. The selective HDAC inhibitors have been considered to be a critical target for the treatment of cardiac disease, especially for ameliorating cardiac dysfunction. In this review, we discuss our current knowledge of the cellular and molecular basis of HDACs in mediating cardiac development and hypertrophy and related pharmacologic interventions in heart disease.

Restenosis after angioplasty of peripheral arteries is a clinical problem involving oxidative stress. Hydrogen sulfide (H₂S) participates in oxidative stress regulation and activates nuclear factor erythroid 2-related factor 2 (Nrf2). This study investigated the effect of H₂S and Nrf2 on restenosis-induced arterial injury. Using an in vivo rat model of restenosis, we investigated whether H₂S inhibits restenosis after percutaneous transluminal angioplasty (PTA) and the oxidative stress-related mechanisms implicated therein. The involvement of Nrf2 was explored using Nrf2-shRNA. Neointimal formation and the deposition of elastic fibers were assessed histologically. Inflammatory cytokine secretion and the expression of proteins associated with oxidative stress and inflammation were evaluated. The artery of rats subjected to restenosis showed increased arterial intimal thickness, with prominent elastic fiber deposition. Sodium hydrosulfide (NaHS), an H₂S donor, counteracted these changes in vivo. Restenosis caused a decrease in anti-oxidative stress signaling. This phenomenon was inhibited by NaHS, but Nrf2-shRNA counteracted the effects of NaHS. In terms of inflammation, inflammatory cytokines were upregulated, whereas NaHS suppressed the induced inflammatory reaction. Similarly, Nrf2 downregulation blocked the effect of NaHS. In vitro studies using aortic endothelial and vascular smooth muscle cells isolated from experimental animals showed consistent results as those of in vivo studies, and the participation of the nuclear factor-kappa B signaling pathway was demonstrated. Collectively, H₂S played a role in regulating post-PTA restenosis by alleviating oxidative stress, modulating anti-oxidant defense, and targeting Nrf2-related pathways via nuclear factor-kappa B signaling.

Degeneration of photoreceptors is a major cause of blindness. Identifying new methods for the generation of photoreceptors offers valuable options for a cell replacement therapy of blindness. Here, we show that primary adult human retinal pigmented epithelium (hRPE) cells were directly converted to postmitotic neurons with various properties of photoreceptors by the neurogenic transcription factor ASCL1 and microRNA124. At Day 8 after the induction of ASCL1 and miRNA124 expression in hRPE cells, 91% of all cells were Tuj1⁺, and 83% of all cells were MAP2⁺ neurons. The cone photoreceptor marker L/M-opsin, the rod photoreceptor marker rhodopsin, and the generic photoreceptor marker recoverin were expressed in 76%, 86%, and 92% of all cells, respectively. Real-time quantitative PCR measurements showed significant and continuous increases in the expression of photoreceptor markers phosducin and recoverin, rod cell markers phosphodiesterases 6 b and arrestin S-antigen, and cone cell markers L/M-opsin and S-opsin in three independent lines of primary hRPE cells at different days of transdifferentiation. Transmission electron microscopy of converted neurons showed disc-like structures similar to those found in photoreceptors. While the converted neurons had voltage-dependent Na⁺, K⁺, and Ca²⁺ currents, light-induced change in membrane potential was not detected. The study demonstrates the feasibility of rapid and efficient transdifferentiation of adult hPRE cells to neurons with many properties of photoreceptors. It opens up a new possibility in cell replacement therapy of blindness caused by photoreceptor degeneration.