Peritoneal dialysis (PD) is an effective renal replacement therapy, but a significant proportion of patients suffer PD-related complications, which limit the treatment duration. Mesothelial-to-mesenchymal transition (MMT) contributes to the PD-related peritoneal dysfunction. We analyzed the genetic reprograming of MMT to identify new biomarkers that may be tested in PD-patients. Microarray analysis revealed a partial overlapping between MMT induced in vitro and ex vivo in effluent-derived mesothelial cells, and that MMT is mainly a repression process being higher the number of genes that are down-regulated than those that are induced. Cellular morphology and number of altered genes showed that MMT ex vivo could be subdivided into two stages: early/epithelioid and advanced/non-epithelioid. RT-PCR array analysis demonstrated that a number of genes differentially expressed in effluent-derived non-epithelioid cells also showed significant differential expression when comparing standard versus low-GDP PD fluids. Thrombospondin-1 (TSP1), collagen-13 (COL13), vascular endothelial growth factor A (VEGFA), and gremlin-1 (GREM1) were measured in PD effluents, and except GREM1, showed significant differences between early and advanced stages of MMT, and their expression was associated with a high peritoneal transport status. The results establish a proof of concept about the feasibility of measuring MMT-associated secreted protein levels as potential biomarkers in PD.

Thrombospondin 1 (TSP1) is a multifunctional matricellular protein. Recent studies demonstrate that TSP1 is highly expressed in adipose tissue (AT) and positively associated with AT inflammation and insulin resistance (IR). In this study, the contribution of different cellular sources of TSP1 to obesity-induced metabolic complications is determined by using mice with either adipocyte or myeloid/macrophage-specific deletion of TSP1 in a diet-induced obese model. The results demonstrated that neither adipocyte nor myeloid/macrophage-specific deletion of TSP1 affected the development of long-term high fat diet (HFD)-induced obesity. Adipocyte-specific deletion of TSP1 did not protect mice from obesity-induced inflammation and IR. On the contrary, obese mice with myeloid/macrophage loss of TSP1 had reduced macrophage accumulation in AT, which was accompanied with reduced inflammation and improved glucose tolerance and insulin sensitivity comparing to obese control mice. Reduced macrophage derived-TGF-β1 signaling and adipose tissue fibrosis were also observed in long-term HF-fed mice with myeloid/macrophage-specific TSP1 deletion. Moreover, in vitro experiments demonstrated an autocrine effect of TSP1-mediated TGF-β activation in macrophages in obesity. Collectively this study highlights the critical contribution of myeloid/macrophage-derived TSP1 to obesity-associated chronic inflammation and IR, which may serve as a new therapeutic target for metabolic disease.

CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148. TSP1 has multiple structural elements and interacts with various cell surface receptors that exhibit differing effects. In order to create the CD148-specific TSP1 fragment, here we investigated the CD148-interacting region in TSP1 using a series of TSP1 fragments and biochemical and biological assays. Our results demonstrate that: 1) CD148 binds to the 1st type 1 repeat in TSP1; 2) Trimeric TSP1 fragments that contain the 1st type repeat inhibit cell proliferation in A431D cells that stably express wild-type CD148 (A431D/CD148wt cells), while they show no effects in A431D cells that lack CD148 or express a catalytically inactive form of CD148. The anti-proliferative effect of the TSP1 fragment in A431D/CD148wt cells was largely abolished by CD148 knockdown and antagonized by the 1st, but not the 2nd and 3rd, type 1 repeat fragment. Furthermore, the trimeric TSP1 fragments containing the 1st type repeat increased the catalytic activity of CD148 and reduced phospho-tyrosine contents of EGFR and ERK1/2, defined CD148 substrates. These effects were not observed in the TSP1 fragments that lack the 1st type 1 repeat. Last, we demonstrate that the trimeric TSP1 fragment containing the 1st type 1 repeat inhibits endothelial cell proliferation in culture and angiogenesis in vivo. These effects were largely abolished by CD148 knockdown or deficiency. Collectively, these findings indicate that the 1st type 1 repeat interacts with CD148, reducing growth factor signals and inhibiting epithelial or endothelial cell proliferation and angiogenesis.

An aberrant systemic artery supply results in recurrent infections in the abnormal lung lobe of intralobar pulmonary sequestration (ILS). The mechanisms underlying such persistent inflammation are unknown. Here, we hypothesize that alteration of an endothelial cell niche for alveolar epithelial cells results in the impaired proliferation potential of alveolar progenitor cells, leading to the defective defense mechanism in intralobar pulmonary sequestration. Paraffin sections of lung tissues from patients with intralobar pulmonary sequestration or from healthy controls were collected for analysis of alveolar epithelial alterations in intralobar pulmonary sequestration by quantitative RT-PCR or immunofluorescent staining. Differential transcripts were identified between human pulmonary artery endothelial cells and human aortic endothelial cells by microarray. Validation of microarray data by quantitative PCR analysis indicated that thrombospondin-1 expression level is low in near-lesion part but high in lesion part of ILS lobe as compared to healthy controls. In vitro 3-D matrigel culture was adopted to evaluate the regulation of alveolar progenitor cells by thrombospondin-1 and CD36. We found that the proliferative potential of alveolar type 2 stem/progenitor cells was impaired in intralobar pulmonary sequestration. Mechanistically, we discovered that endothelial thrombospondin-1 promotes alveolar type 2 cell proliferation through the interaction with CD36. These data demonstrate that alveolar stem cells are impaired in the abnormal lobe from patients with intralobar pulmonary sequestration and imply that restoring epithelial integrity can be beneficial for the future treatments of recurrent infections in lung pathologies.

Diagnosis of Ovarian cancer (OC) has been a challenge, the purpose, therefore is to identify plasma proteins differentially expressed in epithelial ovarian cancer patients. Human plasma samples from patients with OC (n = 138), benign tumors (n = 20) and controls (n = 238) were used. Tandem Mass Tag (TMT) based quantitative analysis by high resolution mass spectrometry, was followed by validation using Quantibody array and ELISA techniques. 507 plasma proteins showed differential protein levels in OC plasma samples. 21 proteins were validated using Quantibody array. Further, nine proteins (CA125, CFD, CST3, ICAM1, IGFBP2, IGFBP3, SPP1, TSP1 and VEGFA) which showed significant differences in protein levels in Quantibody array analysis were validated using ELISA. In ELISA, the levels of CA125, IGFBP2, ICAM1 and SPP1 were significantly increased and levels of Adipsin and TSP1 were decreased in tumors compared to controls and benign group. Epithelial ovarian cancer diagnosis model combining five markers (CA125, IGFBP2, SPP1, TSP1 and ADI) showed 90.24% sensitivity and 94.87% specificity. In conclusion a panel of 5 plasma proteins has been found to be useful in distinguishing plasma samples from epithelial ovarian cancers from patients with benign tumors and healthy normal subjects. This has the potential as a diagnostic assay for epithelial ovarian cancer. SIGNIFICANCE: The significance of this case-control study is based on the large and well defined ovarian cancer patient population (epithelial ovarian cancers including serous and mucinous subtypes), age matched controls and benign ovarian tumors. This study incorporates a discovery phase involving quantitative proteomic analysis of immune-depleted plasma followed by two levels of validation studies involving a selected list of proteins using antibody arrays and ELISA. The validations were performed on an independent set of samples comprising of epithelial ovarian cancer subtypes, controls and benign tumors. The multiple marker combination comprising of Adipsin, CA125, IGFBP2, SPP1 and TSP1 identified in the study by ELISA could enable rapid translation to a larger screening study.

Keywords: CA125; ELISA; IGFBP2; Ovarian cancer; Quantibody array; SPP1; TMT; TSP1.

Thrombospondins (TSPs) are cell adhesion molecules that play an important role in the maintenance of hearing and afferent synaptic connections. Based on their reported function in restoring synaptic connections after stroke, we tested a potential role for TSP1 and TSP2 genes in repairing cochlear synapses following noise injury. We observed a tonotopic gradient in the expression of TSP1 and TSP2 mRNA in control mouse cochleae and an upregulation of these genes following noise exposure. Examining the functional sequelae of these changes revealed that afferent synaptic counts and auditory brainstem responses (ABRs) in noise-exposed TSP1 and TSP2 knockout (-/-) mice exhibited a worst recovery when compared to controls. Consistent with their tonotopic expression, TSP1-/- mice showed greater susceptibility to noise-induced hearing loss (NIHL) at 8 kHz and 16 kHz frequencies, whereas NIHL in TSP2-/- mice occurred only at mid and high frequencies. Further analysis of the ABR waveforms indicated peripheral neuronal damage in TSP2-/- but not in TSP1-/- mice. Noise trauma affecting mid to high frequencies triggered severe seizures in the TSP2-/- mice. We found that decreased susceptibility to audiogenic seizures in TSP1-/- mice was correlated with increased TSP2 protein levels in their inner ears, suggesting that TSP2 might functionally compensate for the loss of TSP1 in these mice. Our data indicate that TSP1 and TSP2 are both involved in susceptibility to NIHL, with TSP2 playing a more prominent role.

OBJECTIVE

To compare the difference in gene expression profiles between parental cell line and drug resistant cell line (CNE-1 and CNE-1/taxol) pre-treated or treated by drugs, and search for genes related to taxol resistance and reversal of taxol resistance phenotype.

METHODS

cDNA microarray was used to detect the difference in gene expression profiles between 6 groups of cells. Combination of multiple filtering genes and detailed analysis of documented resistance genes were used to analyze the data.

RESULTS

Through multiple filtering, 297 differentially expressed genes were screened. The expression of 17 genes was increased or decreased more than 5 folds in CNE-1/taxol compared with CNE-1.Through analyzing documented drug-resistant genes, MDR1 expression was not detected in each group. CYP1A1, one of P450 family members, was not expressed in CNE-1, but significantly increased expressions was found in CNE-1/taxol and these increased expressions were restored by cisplatin. The expression level of some members of tumor necrosis factor family was decreased in CNE-1/taxol and restored by cisplatin, including TNFAIP1, 3 and TNFRSF12A, 21. The differentially expressed members in the caspase family were caspase-4 and caspase-6. The expression of β-tubulin II was down-regulated in CNE-1/taxol. TSP1 was obviously down-regulated in CNE- 1/taxol compared with CNE-1, and a more significant down-regulation of TSP1 was found when treated by taxol. However, it was greatly up-regulated after cisplatin treatment in CNE-1/taxol.

CONCLUSIONS

Some genes are probably related to taxol resistance and reversal of taxol resistance in NPC cells: 297 differentially expressed genes detected by multiple filing, CYP1A1, some members of TNF family and another 17 genes whose differential expression is more than 5 folds between parental cell line and drug resistant cell line. Combination of multiple filtering genes and detailed analysis of documented resistance genes is a good method to study drug resistance and reversal of drug resistance in carcinoma cells.

Introduction: Aging-dependent decline in the angiogenesis of heart is a risk factor for cardiovascular disease. This study was aimed to characterize effect of exercise on angiogenesis alterations and molecular mediators which are related to angiogenesis in the heart under aging condition. Methods: Twenty-one male Wistar rats were assigned into three groups: young, aged, and exercise. Aged animals in the exercise group run on treadmill for 8 weeks. At the end, heart samples were collected and used for histological evaluation , determination of angiogenesis by immunostaining for PECAM-1/ CD31 and expressions of vascular endothelial growth factor-A (VEGF-A), thrombospondin-1 (TSP-1) and nuclear factor kappa B (NF-κB) levels by ELISA. P<0.05 is considered as statistically significant. Results: Our results showed that angiogenesis, and VEGF-A levels were significantly decreased, TSP1 (P >0.0001) and p-NF-κB (P >0.001) levels were significantly increased in the heart of aged group compared to young group. Exercise group showed significant increase in angiogenesis, VEGF-A (P >0.0001), and p-NF-κB (P >0.001) and showed significant decrease in TSP-1 levels (P >0.001) compared to aged group. Moreover, compared to the young group, aged group showed histological changes in the heart, such as interstitial edema, and congestion, whereas, treatment with exercise improved these undesirable changes in the heart of exercise groups. Conclusion: These findings indicated that aging-related decrease in angiogenesis in the heart may mediated by downexpression of VEGF-A and overexpression of TSP-1 proteins. Also, we showed that p-NF-κB protein was increased in the heart of aged rats, this probably mediated by compensatory mechanism. It was also showed that exercise as novel non-pharmacological therapy modifies VEGF-A and TSP-1 and increases p-NF-κB protein levels in the aged heart.

Keywords: Aging; Angiogenesis; Exercise; Heart.

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment (TME) and an important contributor to cancer progression and therapeutic resistance. Regulation of CAF activation is a promising strategy to influence cancer outcomes. Here, we report that ovarian cancer cells (OCs) and TME cells promote the activation of ovarian CAFs, whereas gold nanoparticles (GNPs) of 20 nm in diameter inhibit the activation, as demonstrated by the changes in cell morphology, migration, and molecular markers. GNPs exert the effect by altering the levels of multiple fibroblast activation or inactivation proteins, such as TGF-β1, PDGF, uPA and TSP1, secreted by OCs and TME cells. Thus, GNPs represent a potential tool to help understand multicellular communications existing in the TME as well as devise strategies to disrupt the communication.

Keywords: Cancer-associated fibroblast (CAF); Fibroblast activation; Gold nanoparticle (GNP); Migration; Morphology; Tumor microenvironment (TME).

The regulation of corpus luteus (CL) luteolysis is a complex process involving a myriad of factors. Previously, we have shown the involvement of Nodal in functional luteolysis in mares. Presently, we ask the extent of which Nodal mediation of luteolysis is done through regulation of angioregression. We demonstrated the interaction between Nodal and hypoxia-inducible factor 1 α (HIF1α) and thrombospondin 1/thrombospondin receptor (TSP1/CD36) systems, could mediate angioregression during luteolysis. First, we demonstrated the inhibitory effect of Nodal on the vascular marker platelet/endothelial cell adhesion molecule 1 (CD31). Also, treatment of mid CL explants with vascular endothelial growth factor A (VEGFA) showed a trend on activin-like kinase 7 (Alk7) protein inhibition. Next, Nodal was also shown to activate HIF1α and in vitro culture of mid CL explants under decreased oxygen level promoted Nodal expression and SMAD family member 3 (Smad3) phosphorylation. In another experiment, the crosstalk between Nodal and TSP1/CD36 was investigated. Indeed, Nodal increased the expression of the anti-angiogenic TSP1 and its receptor CD36 in mid CL explants. Finally, the supportive effect of prostaglandin F2α (PGF2α) on TSP1/CD36 was blocked by SB431542 (SB), a pharmacological inhibitor of Nodal signaling. Thus, we evidenced for the first time the in vitro interaction between Nodal and both HIF1α and TSP1 systems, two conserved pathways previously shown to be involved in vascular regression during luteolysis. Considering the given increased expression of Nodal in mid CL and its role on functional luteolysis, the current results suggest the additional involvement of Nodal in angioregression during luteolysis in the mare, particularly in the activation of HIF1α and TSP1/CD36.

Our previous research implied mouse skin-derived precursors (mSKPs) possessed the capacity of anti-ultraviolet B (UVB) irradiation damage, and the mechanisms might be associated with transforming growth factor-β (TGF-β) signaling pathway activation. In this study, we investigated and compared the response to UVB irradiation between mSKPs and dermal mesenchymal stem cells (dMSCs), and explored the underlying mechanisms. Irradiation damage such as decreased cell viability, cell senescence, and cell death was observed in both mSKPs and dMSCs at 24 h after UVB exposure. In mSKPs, change in cell morphology, viability, cell senescence and death at the following time points implied the recovery of UVB irradiation damage. Additionally, thrombospondin1 (TSP1) and TGF-β1 increased significantly in mSKPs' supernatant after UVB irradiation. The gene expression of TSP1, TGF-β1, metalloproteinase 1 (MMP1), and Collagen I elevated shortly after the UVB exposure. The protein expression of TSP1, TGF-β1, MMP1, Collagen I, smad2/3, and p-smad2/3 at multiple time points after the UVB exposure was consistent with the gene expression results. In dMSCs, no obvious recovery was noticed. Together, these results revealed that in mSKPs, one of the mechanisms to attenuate the UVB irradiation damage might be the early activation of TGF-β/Smad pathway by TSP1. Given that mSKPs could differentiate into fibroblast-like SKP-derived fibroblasts (SFBs) in vivo or with the presence of serum, mSKPs might serve as a therapeutic potential for fibroblasts supplement and UVB irradiation damage treatment.Abbreviations: SKPs: skin-derived precursors; mSKPs: mouse SKPs; UVB: ultraviolet B; TGF-β/Smad: transforming growth factor-β/Smad; TSP1: thrombospondin 1; MMP 13: metalloproteinases 13; TβRII: TGF-β receptor II; SFBs: SKP-derived fibroblasts; KEGG: Kyoto encyclopedia of genes and genomes; DEGs: differentially expressed genes; dMSCs: dermal mesenchymal stem cells; LM: light microscope; CCK-8: cell counting kit 8; ELISA: Enzyme-linked immuno sorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; TSPs: thrombospondins; ECM: extracellular matrix; R-smads: receptor-regulated smads.

Bacteriophages (phages) are widely used as biocontrol agents in food and as antibacterial agents for treatment of food production plant surfaces. An important feature of such phages is broad infectivity towards a given pathogenic species. Phages attach to the surfaces of bacterial cells using receptor binding proteins (RBPs), namely tail fibers or tailspikes (TSPs). The binding range of RBPs is the primary determinant of phage host range and infectivity, and therefore dictates a phage's suitability as an antibacterial agent. Phages EP75 and EP335 broadly infect strains of E. coli serotype O157. To better understand host recognition by both phages, here we focused on characterizing the structures and functions of their RBPs. We identified two distinct tail fibers in the genome of the podovirus EP335: gp12 and gp13. Using fluorescence microscopy, we reveal how gp13 recognizes strains of E. coli serotypes O157 and O26. Phage EP75 belongs to the Kuttervirus genus within the Ackermannviridae family and features a four TSP complex (TSPs 1-4) that is universal among such phages. We demonstrate enzymatic activity of TSP1 (gp167) and TSP2 (gp168) toward the O18A and O157 O-antigens of E. coli, respectively, as well as TSP3 activity (gp169.1) against O4, O7, and O9 Salmonella O-antigens. TSPs of EP75 present high similarity to TSPs from E. coli phages CBA120 (TSP2) and HK620 (TSP1) and Salmonella myovirus Det7 (TSP3), which helps explain the cross-genus infectivity observed for EP75.

Keywords: Bacteriophage; Escherichia coli O157; Lipopolysaccharide; O-antigen; Receptor binding protein; STEC; Salmonella; Tail fiber; Tailspike.

Cardiac fibrosis (CF) is regulated by multiple factors, including transforming growth factor β1 (TGFβ1) and non-coding RNAs. Thrombospondin 1 (TSP1) is a physiologic regulator of TGFβ activation. Here, we performed microarray analyses on mRNAs and lncRNAs differentially-expressed in the CF and normal rat hearts. KEGG signaling annotation and GO enrichment analyses were performed to validate the roles of extracellular matrix (ECM) and TSP1-enhanced TGFβ activation in CF. The co-expression network between differentially-expressed lncRNAs and ECM-related factors was constructed to identify candidate lncRNAs and miRNAs. We found that lncRNA Homo sapiens ring finger protein 7 (lnc RNF7) was significantly correlated with TSP1 and ECM. Lnc RNF7 silence could attenuate isoproterenol (ISP)-induced CF in rat heart in vivo and in rat cardiac fibroblasts in vitro. Moreover, angiotensin II (Ang II) -induced CF in rat cardiac fibroblasts could also be attenuated by Lnc RNF7 silence. Furthermore, miR-543 could simultaneously target lnc RNF7 and 3' UTR of TSP1. Lnc RNF7 silence suppressed, while miR-543 inhibition promoted TSP1 protein and TGFβ activation, as well as ECM markers expression. The effects of lnc RNF7 silence was significantly reversed by miR-543 inhibition. In conclusion, CF progression might be regulated by lnc RNF7/miR-543 axis via TSP1-mediated TGFβ activation.

Most therapeutic attempts to prevent the progression of kidney diseases have been based on interventions to inhibit the production of transforming growth factor-β (TGF-β). Thrombospondins (TSPs) play an important role in activating TGF-β. In the healthy kidney, two TSPs are expressed, TSP1 and TSP2, which exert contrasting effects. While TSP1 is a major activator of TGF-β in renal cells and exerts pro-inflammatory effects both in vitro and in vivo, TSP2 lacks the ability for TGF-β activation but regulates matrix remodeling and inflammation in experimental kidney disease. The effects of TSPs in the kidney have been mostly investigated by using the murine model of unilateral ureteral obstruction. In this model, TSP1 expression is increased along with the development of interstitial fibrosis and TGF-β. Relief of the obstruction gradually improves renal function and decreases the expression in TSP1 and TGF-β1. Several inhibitors of TSP1 prevented progressive interstitial fibrosis in murine models of ureteral obstruction, suggesting that control of latent TGF-β activation by inhibiting TSP1 might represent a novel potential target for preventing renal interstitial fibrosis. However, further studies are needed to assess whether TSP1-mediated TGF-β activation can be safely used in humans. In fact, TSPs normally act to suppress tumors in vivo. Moreover, TGF-β can exert a pivotal function in the immune system, as it may induce the production of regulatory T cells and suppress B cell responses. Knowledge of the molecular mechanisms involved in TGF-β regulation may help in finding effective treatments of tissue fibrosis, cancer and autoimmune disease.

Numerous age-dependent alterations at the molecular, cellular, tissue and organ systems levels underlie the pathophysiology of aging. Herein, the focus is upon the secreted protein thrombospondin-1 (TSP1) as a promoter of aging and age-related diseases. TSP1 has several physiological functions in youth including promoting neural synapse formation, mediating responses to ischemic and genotoxic stress, minimizing hemorrhage, limiting angiogenesis, and supporting wound healing. These acute functions of TSP1 generally require only transient expression of the protein. However, accumulating basic and clinical data reinforce the view that chronic diseases of aging are associated with accumulation of TSP1 in the extracellular matrix, which is a significant maladaptive contributor to the aging process. Identification of the relevant cell types that chronically produce and respond to this TSP1 and the molecular mechanisms that mediate the resulting maladaptive responses could direct the development of therapeutic agents to delay or revert age-associated maladies.

Recent advances provide evidence that the cellular signalling pathway comprising the ligand-receptor duo of thrombospondin-1 (TSP1) and CD47 is involved in mediating a range of diseases affecting renal, vascular, and metabolic function, as well as cancer. In several instances, research has barely progressed past pre-clinical animal models of disease and early phase 1 clinical trials, while for cancers, anti-CD47 therapy has emerged from phase 2 clinical trials in humans as a crucial adjuvant therapeutic agent. This has important implications for interventions that seek to capitalize on targeting this pathway in diseases where TSP1 and/or CD47 play a role. Despite substantial progress made in our understanding of this pathway in malignant and cardiovascular disease, knowledge and translational gaps remain regarding the role of this pathway in kidney and metabolic diseases, limiting identification of putative drug targets and development of effective treatments. This review considers recent advances reported in the field of TSP1-CD47 signalling, focusing on several aspects including enzymatic production, receptor function, interacting partners, localization of signalling, matrix-cellular and cell-to-cell cross talk. The potential impact that these newly described mechanisms have on health, with a particular focus on renal and metabolic disease, is also discussed.

Keywords: CD47 (IAP); THBS1); ageing; fibrosis; glucose homeostasis; kidney injury; matricellular; reactive oxygen species; thrombospondin-1 (TSP1.

The subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus are known as neurogenic niches. We show that the median eminence (ME) of the hypothalamus comprises BrdU⁺ newly proliferating cells co-expressing NG2 (oligodendrocyte progenitors) and RIP (pre-myelinating oligodendrocytes), suggesting their differentiation toward mature oligodendrocytes (OLs). ME cells can generate neurospheres (NS) in vitro, which differentiate mostly to OLs compared with SVZ-NS that typically generate neurons. Interestingly, this population of oligodendrocyte progenitors is increased in the ME from experimental autoimmune encephalomyelitis (EAE)-affected mice. Notably, the thrombospondin 1 (TSP1) expressed by astrocytes, acts as negative regulator of oligodendrogenesis in vitro and is downregulated in the ME of EAE mice. Importantly, transplanted ME-NS preferentially differentiate to MBP⁺ OLs compared with SVZ-NS in Shiverer mice. Hence, discovering the ME as a new site for myelin-producing cells has a great importance for advising future therapy for demyelinating diseases and spinal cord injury.

Research on polycystic ovarian syndrome (PCOS) remains intense due to its evolving impact on metabolism, reproduction and cardiovascular function. Changes in metabolic pathways can also significantly impact renal function including the development of Focal Segmental Glomerulosclerosis (FSGS), one of the most highly investigated renal diseases. In FSGS, scarring of the glomerulus vascular tuft damages the kidneys. Onset of FSGS may either be congenital or due to other disorders that affect the metabolism and normal kidney function. Both PCOS and FSGS appear to be associated with Transforming Growth Factor-β (TGF-β) signalling. Over-expression of TGF-β may be due to the activation of the thrombospondin 1 (TSP1) gene, which increases the probability of developing renal disorders. Higher androgen levels in PCOS may also cause podocyte damage thus directly impacting development of FSGS. This article reviews the role of TGF-β's in PCOS and FSGS and explores the inter-relationship between these two disorders.

Keywords: Activin; Focal Segmental Glomerulosclerosis; Polycystic ovarian syndrome; Thrombospondin-1; Transforming Growth Factor-β.

Objective: Postnatal angiogenesis is critical in vascular homeostasis and repair. m⁶A RNA methylation is emerging as a new layer for fine-tuning gene expression. Although the contribution of the m⁶A-catalyzing enzyme, METTL3 (methyltransferase-like 3), in cancer biology has been described, its role in endothelial cell (EC) function, particularly during angiogenesis, remains unclear. Approach and Results: To characterize the relevance of METTL3 in angiogenesis regulation, we performed gain- and loss-of-function studies in vitro. We demonstrated that depletion of METTL3 in ECs reduced the level of m⁶A and impaired EC function, whereas adenovirus-mediated METTL3 overexpression increased angiogenesis. Mechanistically, we showed that METTL3 depletion in ECs decreased mature angiogenic microRNAs let-7e-5p and the miR-17-92 cluster, and increased the expression of their common target, Tsp1 (thrombospondin 1). Conversely, Ad.METTL3 increased the expression of let-7e-5p and miR-17-92 cluster and reduced protein levels of Tsp1 in ECs. Moreover, overexpression of let-7e-5p and miR-18a-5p restored the angiogenic potential of METTL3-depleted ECs. We corroborated our data in vivo employing 3 mouse models. When tested in an in vivo Matrigel plug assay, METTL3-depleted ECs had diminished ability to vascularize the plug, whereas overexpression of METTL3 promoted angiogenesis. Local Ad.METTL3 gene transfer increased postischemic neovascularization in mice with either unilateral limb ischemia or myocardial infarction.

Conclusions: METTL3 regulates m⁶A RNA methylation in ECs. Endogenous METTL3 is essential for EC function and angiogenesis, potentially through influencing let-7e and miR-17-92 cluster processing. Thus, the therapeutic modulation of METTL3 should be considered as a new approach for controlling angiogenic responses in the clinical setting.

Keywords: endothelial cell; gene expression; homeostasis; microRNA; thrombospondin.

Inhibitor of DNA binding (Id) proteins play important roles in regulating cardiac development via paracrine signaling. Id1/Id3 knockout mice die at mid-gestation with multiple cardiac defects. Single Id knockout studies have not reported cardiomyopathies. To bypass embryonic lethality we used Tie2CRE-mediated recombination to conditionally delete Id1 against global Id3 ablation (Id cDKOs), which develops adult-onset dilated cardiomyopathy. We confirm upregulation of thrombospondin-1 (TSP1) in Id cDKO hearts. Colocalization studies reveal increased TSP1 expression in the vicinity of endothelial cells and near regions of endocardial fibrosis/disruption. Downstream fibrotic molecules were upregulated. Endocardial capillary density was reduced with evidence of vascular distention. Treatment of Id cDKO cardiac explants with LSKL, a peptide antagonist of TSP1 activation of TGFβ, reversed the increased expression of fibrotic molecules. We conducted bone marrow transplant experiments in which we transferred bone marrow cells from Id cDKO mice into lethally irradiated WT mice. The majority of WT recipients of Id cDKO bone marrow cells phenocopied Id cDKO cardiac fibrosis 4 months post-transplantation. Injection of LSKL into adult Id cDKO mice led to downregulation of fibrotic molecules. The results prompt caution when bone marrow transfers from individuals potentially carrying mutations in the Id axis are applied in clinical settings.