Gonorrhea prevalence in adults in Brazil: Spectrum-STI trend estimation, 2000-2020

ABSTRACT Background: Gonorrhea is not a notifiable disease in Brazil, and the national health information system does not collect data on reported cases or infection prevalence. Methods: We compiled published data on gonorrhea prevalence in Brazil from cross-sectional surveys and clinical trials between 2000 and 2020. The study entry criteria included a sample size of 50 or more, and Neisseria gonorrhoeae infection detected in urine, urethral, anal, or cervicovaginal specimens using either Nucleic Acid Amplification Test or culture. Gonorrhea prevalence trends between 2000 and 2020 were generated using Spectrum-STI, a statistical trend-fitting model. Results: Forty-five studies with 59 gonorrhea prevalence data points were identified. Fifty data points were for women and represented 21,815 individuals, eight for men encompassing a total of 4,587 individuals, and one for transgender people comprising 345 individuals. The Spectrum-STI estimate for the prevalence of urogenital infection with gonorrhea in women 15-49 in 2020 was 0.63% (95% confidence interval (CI): 0.13-2.23) and was lower than the 1.05% estimated value for 2000 (95% CI: 0.36-2.79). The corresponding figures for men were 0.70% (95% CI: 0.16-2.44) and 1.14% (95% CI: 0.34-3.15). Anal prevalence estimates could not be generated because of insufficient data (three data points). Conclusions: These results suggest that the overall prevalence of genitourinary gonococcal infections in Brazil is less than 1%. Data on gonorrhea prevalence in men and in populations at increased STI vulnerability are limited.

Exploring the potential relationship between collagen cross-linking and impaired myocardial relaxation in Marfan syndrome: An observational study using serum biomarkers.

BACKGROUND: Increased collagen cross-linking (CCL) has been described in hypertensive cardiomyopathy by means of reduced serum ratio of serum carboxyterminal telopeptide of collagen type I (CITP) to matrix metalloproteinase-1 (MMP1). Previous studies have demonstrated the existence of primary impaired diastole in patients with Marfan syndrome (MFS), but little is known about the pathophysiology of this condition. METHODS: 60 MFS patients (without previous cardiovascular surgery or significant valvular regurgitation) and 24 healthy controls (age and sex-matched) were enrolled. All participants underwent a comprehensive transthoracic echocardiographic study, including left atrial and left ventricular speckle-tracking strain analysis. CITP and MMP1 were measured in peripheral blood. RESULTS: All participants had normal diastolic function according to guidelines. Peak left atrial strain in the reservoir phase (LASr) was significantly reduced in the MFS cohort compared to controls (32.2 ± 9.4 vs 43.9 ± 7.0%; p < 0.001). Serum CITP and CITP:MMP1 ratio were lower among MFS patients, showing significant correlations with LASr (R = 0.311; p = 0.020 and R = 0.437; p = 0.001, respectively). The MFS cohort was divided into quartiles of LASr. MFS patients in the lowest quartile of LASr (<26%) had significantly lower values of CITP:MMP1 ratio compared to the other quartiles. CONCLUSIONS: The analysis of serum biomarkers revealed the presence of increased CCL in association with reduced LASr in the MFS cohort. Our results suggest that excessive CCL may play a role in the development of primary myocardial impairment in these patients. Future studies are needed to confirm our findings and evaluate the prognostic role of CCL markers in the MFS population.

The challenge of determining lysyl oxidase activity: Old methods and novel approaches.

The lysyl oxidase (LOX) family of enzymes catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-linkages, an essential process for extracellular matrix (ECM) maturation. Elevated LOX expression levels leading to increased LOX activity is associated with diverse pathologies including fibrosis, cancer, and cardiovascular diseases. Different protocols have been so far established to detect and quantify LOX activity from tissue samples and cultured cells, all of them showing advantages and drawbacks. This review article presents a critical overview of the main features of currently available methods as well as introduces some recent technologies called to revolutionize our approach to LOX catalysis.

Boosting collagen deposition with a lysyl oxidase/bone morphogenetic protein-1 cocktail.

This book chapter describes the use of exogenous application of lysyl oxidase (LOX) and bone morphogenetic protein-1 (BMP1) to enhance collagen synthesis and deposition from fibroblasts in culture. The protocol includes the generation of human embryonic kidney (HEK) 293 cell lines overexpressing human LOX and BMP1 constructs in order to obtain supernatants enriched in these factors. Incubation of fibroblast monolayers with these conditioned media strongly increases the capacity of these cells to deposit collagen onto the insoluble extracellular matrix. We also describe the use of these decellularized fibroblast-derived matrices as a substrate for the growth and differentiation of mesenchymal stem cells.

Validação de uma escala para a análise das percepções dos moradores sobre o impacto social de um centro esportivo / Validación de una escala para el análisis de las percepciones de los residentes sobre el impacto social de un centro deportivo / Validation of a scale to analyse resident's perceptions of the social impact of a sports centre

O objetivo deste estudo foi validar uma escala para a análise das percepções dos moradores sobre o impacto social de um centro esportivo. Se recolheu uma amostra de 406 moradores do município de Moncada (Valência) com erro de amostragem de ± 4,82. Os moradores responderam a um questionário de 31 itens que incluíram possíveis impactos sociais derivados da presença do complexo esportivo na localidade. A aplicação da análise fatorial exploratória e confirmatória reduziu a escala para 28 indicadores distribuídos em sete dimensões de impacto: impacto sociocultural, impacto socioeconômico, impacto na imagem e promoção do município, impacto no desenvolvimento e infraestruturas urbanas, impacto na coesão social, igualdade e equidade, impacto na saúde e impacto nos hábitos e níveis de atividade física. Os resultados permitiram verificar a validade e confiabilidade da escala proposta para o objeto de estudo

La finalidad de este estudio es validar una escala para el análisis de las percepciones de los residentes sobre el impacto social de un centro deportivo. Se recogió una muestra de 406 residentes del municipio de Moncada (Valencia), con un error de muestreo de ±4,82, que contestaron a una encuesta de 31 ítems que recogían posibles impactos sociales derivados de la presencia del centro deportivo en la localidad. La aplicación de análisis factorial exploratorio y confirmatorio redujo la escala a 28 indicadores distribuidos en siete dimensiones de impacto: impacto sociocultural, impacto socioeconómico, impacto en la imagen y la promoción del municipio, impacto en el desarrollo urbano y las infraestructuras, impacto en la cohesión social, igualdad y equidad, impacto sobre la salud e impacto sobre los hábitos y niveles de actividad física. Los resultados permitieron comprobar la validez y fiabilidad de la escala propuesta para el objeto de estudio

The purpose of this study is to validate a scale to analyse residents' perceptions of the social impact of a sports centre. A sample of 406 residents of Moncada, Valencia, was collected with sampling error of ±4.82. They responded to a 31-item survey on potential social impacts of the presence of the sports centre in that town. Exploratory and confirmatory factorial analysis reduced the scale to 28 indicators distributed in seven impact dimensions: sociocultural; socioeconomic; image and promotion of the town; urban development and infrastructures; social cohesion; equality and equity; health; and physical activity habits and levels. The results made it possible to check the validity and reliability of the scale proposed for the object of study

A hierarchical network of hypoxia-inducible factor and SMAD proteins governs procollagen lysyl hydroxylase 2 induction by hypoxia and transforming growth factor ß1.

Collagens are extracellular matrix (ECM) proteins that support the structural and biomechanical integrity of many tissues. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) encodes the only lysyl hydroxylase (LH) isoform that specifically hydroxylates lysine residues in collagen telopeptides, a post-translational modification required for the formation of stabilized cross-links. PLOD2 expression is induced by hypoxia and transforming growth factor-ß1 (TGF-ß1), well-known stimuli for the formation of a fibrotic ECM, which can lead to pathological fibrosis underlying several diseases. Here, using human and murine fibroblasts, we studied the molecular determinants underlying hypoxia- and TGF-ß1-induced PLOD2 expression and its impact on collagen biosynthesis. Deletion mapping and mutagenesis analysis identified specific binding sites for hypoxia-inducible factors (HIF) and TGF-ß1-activated SMAD proteins on the human PLOD2 gene promoter that were required for these stimuli to induce PLOD2 expression. Interestingly, our experiments also revealed that HIF signaling plays a preponderant role in the SMAD pathway, as intact HIF sites were absolutely required for TGF-ß1 to exert its effect on SMAD-binding sites. We also found that silencing PLOD2 expression did not alter soluble collagen accumulation in the extracellular medium, but it effectively abolished the deposition into the insoluble collagen matrix. Taken together, our findings reveal the existence of a hierarchical relationship between the HIF and SMAD signaling pathways for hypoxia- and TGF-ß1-mediated regulation of PLOD2 expression, a key event in the deposition of collagen into the ECM.

Lysyl Oxidases: Functions and Disorders.

Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize lysyl and hydroxylysyl residues in collagen and elastin, as a first step in the stabilization of these extracellular matrix proteins through the formation of covalent cross-linkages, an essential process for connective tissue maturation. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, being genetically different protein products with a high degree of homology in the catalytic carboxy terminal end and a more variable amino terminal proregion. Intensive investigation in the last years has delineated the main biological functions of these enzymes and their involvement in several pathologies including fibrosis, cancer, and ocular disorders. This review article summarizes the major findings on the role of LOX isoforms, with particular focus on their contribution to the development and progression of human disorders.

Epithelial contribution to the profibrotic stiff microenvironment and myofibroblast population in lung fibrosis.

The contribution of epithelial-to-mesenchymal transition (EMT) to the profibrotic stiff microenvironment and myofibroblast accumulation in pulmonary fibrosis remains unclear. We examined EMT-competent lung epithelial cells and lung fibroblasts from control (fibrosis-free) donors or patients with idiopathic pulmonary fibrosis (IPF), which is a very aggressive fibrotic disorder. Cells were cultured on profibrotic conditions including stiff substrata and TGF-ß1, and analyzed in terms of morphology, stiffness, and expression of EMT/myofibroblast markers and fibrillar collagens. All fibroblasts acquired a robust myofibroblast phenotype on TGF-ß1 stimulation. Yet IPF myofibroblasts exhibited higher stiffness and expression of fibrillar collagens than control fibroblasts, concomitantly with enhanced FAKY397 activity. FAK inhibition was sufficient to decrease fibroblast stiffness and collagen expression, supporting that FAKY397 hyperactivation may underlie the aberrant mechanobiology of IPF fibroblasts. In contrast, cells undergoing EMT failed to reach the values exhibited by IPF myofibroblasts in all parameters examined. Likewise, EMT could be distinguished from nonactivated control fibroblasts, suggesting that EMT does not elicit myofibroblast precursors either. Our data suggest that EMT does not contribute directly to the myofibroblast population, and may contribute to the stiff fibrotic microenvironment through their own stiffness but not their collagen expression. Our results also support that targeting FAKY397 may rescue normal mechanobiology in IPF.

Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with other methods.

BACKGROUND: The discovery of mutations in the epidermal growth factor receptor gene (EGFR) related to the clinical response to tyrosine kinase inhibitors, has transformed the management of non-small cell lung cancer (NSCLC). Several methods have been developed for determination of mutations in EGFR, with different sensitivity and potential ability to detect a different number of mutations. METHODS: We developed a screening method by high resolution melting (HRM) to detect EGFR mutations, and compared the results of 123 fixed in formalin and paraffin embedded (FFPE) tumor tissue samples with the detection of mutations by allele-specific PCR. In samples with discordant results, Sanger and massive parallel sequencing (MPS) were additionally performed. RESULTS: Eight samples showed discordant results between both methods. Three samples with negative results by allele specific PCR and positive by HRM were confirmed by Sanger sequencing (p.S768I+p.V769L, T751_I759del and p.E709K+p.G719A; patients 1, 3 and 4, respectively). One sample with a negative result by HRM, and positive by allele specific PCR (p.T790M; patient 2), was confirmed by Sanger sequencing. Additionally, two positive samples for a deletion in exon 19 by allele-specific PCR, were negative by Sanger sequencing and HRM (patients 2 and 5) and finally, two samples were negative by allele-specific PCR and positive by HRM and Sanger sequencing due to synonymous variants in exon 21. CONCLUSIONS: HRM is a good method for mutational screening in EGFR. It is able to detect any change in the sequence of exons 18-21, providing high cost/effectiveness, but samples with low tumor burden may produce false negatives results.

Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction.

AIMS: After myocardial infarction (MI), extensive remodelling of the extracellular matrix contributes to scar formation. While aiming to preserve tissue integrity, this fibrotic response is also associated with adverse events, including a markedly increased risk of heart failure, ventricular arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross-links. This study investigates the contribution of LOX family members to the heart response to MI. METHODS AND RESULTS: Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1-4) was strongly increased upon MI, and this response was accompanied by a significant accumulation of mature collagen fibres in the infarcted area. LOX expression was observed in areas of extensive remodelling, partially overlapping with α-smooth muscle actin-expressing myofibroblasts. Tumour growth factor-ß as well as hypoxia-activated pathways contributed to the induction of LOX expression in cardiac fibroblasts. Finally, in vivo post-infarction treatment with the broadband LOX inhibitor ß-aminopropionitrile or, selectively, with a neutralizing antibody against the canonical LOX isoform attenuated collagen accumulation and maturation and also resulted in reduced ventricular dilatation and improved cardiac function. CONCLUSION: LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery.

Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.

OBJECTIVE: Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-ß signaling. TGF-ß is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-ß signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. APPROACH AND RESULTS: Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-ß pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. CONCLUSIONS: In Marfan VSMC, both in tissue and in culture, there are variable TGF-ß-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation.

Targeting of Gamma-Glutamyl-Cysteine Ligase by miR-433 Reduces Glutathione Biosynthesis and Promotes TGF-ß-Dependent Fibrogenesis.

AIMS: Glutathione (GSH) is the main antioxidant against cell damage. Several pathological states course with reduced nucleophilic tone and perturbation of redox homeostasis due to changes in the 2GSH/GSSG ratio. Here, we investigated the regulation of the rate-limiting GSH biosynthetic heterodimeric enzyme γ-glutamyl-cysteine ligase (GCL) by microRNAs (miRNAs). RESULTS: "In silico" analysis of the 3'- untranslated regions (UTRs) of both catalytic (GCLc) and regulatory (GCLm) subunits of GCL enabled an identification of miR-433 as a strong candidate for the targeting of GCL. Transitory overexpression of miR-433 in human umbilical vein endothelial cells (HUVEC) showed a downregulation of both GCLc and GCLm in a nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-independent manner. Increases in pro-oxidant stimuli such as exposure to hydrogen peroxide or GSH depletion in endothelial and hepatic cells caused an expected increase in GCLc and GCLm protein expression and abrogation of miR-433 levels, thus supporting a cross-regulation of these pathways. Treatment of HUVEC with miR-433 resulted in reduced antioxidant and redox potentials, increased S-glutathionylation, and reduced endothelial nitric oxide synthase activation. In vivo models of renal and hepatic fibrosis were associated with transforming growth factor ß1 (TGF-ß1)-related reduction of GCLc and GCLm levels that were miR-433 dependent. INNOVATION AND CONCLUSION: We describe for the first time an miRNA, miR-433, capable of directly targeting GCL and promoting functional consequences in endothelial physiology and fibrotic processes by decreasing GSH levels.

A pathogenetic role for endothelin-1 in peritoneal dialysis-associated fibrosis.

In patients undergoing peritoneal dialysis (PD), chronic exposure to nonphysiologic PD fluids elicits low-grade peritoneal inflammation, leading to fibrosis and angiogenesis. Phenotype conversion of mesothelial cells into myofibroblasts, the so-called mesothelial-to-mesenchymal transition (MMT), significantly contributes to the peritoneal dysfunction related to PD. A number of factors have been described to induce MMT in vitro and in vivo, of which TGF-ß1 is probably the most important. The vasoconstrictor peptide endothelin-1 (ET-1) is a transcriptional target of TGF-ß1 and mediates excessive scarring and fibrosis in several tissues. This work studied the contribution of ET-1 to the development of peritoneal damage and failure in a mouse model of PD. ET-1 and its receptors were expressed in the peritoneal membrane and upregulated on PD fluid exposure. Administration of an ET receptor antagonist, either bosentan or macitentan, markedly attenuated PD-induced MMT, fibrosis, angiogenesis, and peritoneal functional decline. Adenovirus-mediated overexpression of ET-1 induced MMT in human mesothelial cells in vitro and promoted the early cellular events associated with peritoneal dysfunction in vivo. Notably, TGF-ß1-blocking peptides prevented these actions of ET-1. Furthermore, a positive reciprocal relationship was observed between ET-1 expression and TGF-ß1 expression in human mesothelial cells. These results strongly support a role for an ET-1/TGF-ß1 axis as an inducer of MMT and subsequent peritoneal damage and fibrosis, and they highlight ET-1 as a potential therapeutic target in the treatment of PD-associated dysfunction.

Matrix stiffening and ß1 integrin drive subtype-specific fibroblast accumulation in lung cancer.

UNLABELLED: The crucial role of tumor-associated fibroblasts (TAF) in cancer progression is now clear in non-small cell lung cancer (NSCLC). However, therapies against TAFs are limited due to a lack of understanding in the subtype-specific mechanisms underlying their accumulation. Here, the mechanical (i.e., matrix rigidity) and soluble mitogenic cues that drive the accumulation of TAFs from major NSCLC subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were dissected. Fibroblasts were cultured on substrata engineered to exhibit normal- or tumor-like stiffnesses at different serum concentrations, and critical regulatory processes were elucidated. In control fibroblasts from nonmalignant tissue, matrix stiffening alone increased fibroblast accumulation, and this mechanical effect was dominant or comparable with that of soluble growth factors up to 0.5% serum. The stimulatory cues of matrix rigidity were driven by ß1 integrin mechano-sensing through FAK (pY397), and were associated with a posttranscriptionally driven rise in ß1 integrin expression. The latter mechano-regulatory circuit was also observed in TAFs but in a subtype-specific fashion, because SCC-TAFs exhibited higher FAK (pY397), ß1 expression, and ERK1/2 (pT202/Y204) than ADC-TAFs. Moreover, matrix stiffening induced a larger TAF accumulation in SCC-TAFs (>50%) compared with ADC-TAFs (10%-20%). In contrast, SCC-TAFs were largely serum desensitized, whereas ADC-TAFs responded to high serum concentration only. These findings provide the first evidence of subtype-specific regulation of NSCLC-TAF accumulation. Furthermore, these data support that therapies aiming to restore normal lung elasticity and/or ß1 integrin-dependent mechano regulation may be effective against SCC-TAFs, whereas inhibiting stromal growth factor signaling may be effective against ADC-TAFs. IMPLICATIONS: This study reveals distinct mechanisms underlying the abnormal accumulation of tumor-supporting fibroblasts in two major subtypes of lung cancer, which will assist the development of personalized therapies against these cells.

HLA-B35 and dsRNA induce endothelin-1 via activation of ATF4 in human microvascular endothelial cells.

Endothelin 1 (ET-1) is a key regulator of vascular homeostasis. We have recently reported that the presence of Human antigen class I, HLA-B35, contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by upregulating ET-1 and proinflammatory genes. Likewise, a Toll-like receptor 3 (TLR3) ligand, Poly(I:C), was shown to induce ET-1 expression in HDMECs. The goal of this study was to determine the molecular mechanism of ET-1 induction by these two agonists. Because HLA-B35 expression correlated with induction of Binding Immunoglobulin Protein (BiP/GRP78) and several heat shock proteins, we first focused on ER stress and unfolded protein response (UPR) as possible mediators of this response. ER stress inducer, Thapsigargin (TG), HLA-B35, and Poly(I:C) induced ET-1 expression with similar potency in HDMECs. TG and HLA-B35 activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of XBP1, but did not affect the ATF6 pathway. Poly(I:C) also activated eIF2α/ATF4 in a protein kinase R (PKR)-dependent manner. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by all three agonists. Additional experiments have demonstrated that the JNK and NF-κB pathways are also required for ET-1 upregulation by these agonists. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-κB complex was increased in the agonist treated cells. The functional role of c-JUN in responses to HLA-B35 and Poly(I:C) was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. The ER stress/UPR and TLR3 pathways converge on eIF2α/ATF4 during activation of endothelial cells.

Adenoviral gene transfer of endothelin-1 in the lung induces pulmonary fibrosis through the activation of focal adhesion kinase.

Endothelin-1 (ET-1) has been implicated in the development of pulmonary fibrosis, based on its capacity in vitro to promote extracellular matrix (ECM) production and contraction, and on studies showing elevated expression of ET-1 and its receptors in patients with pulmonary fibrosis. However, the in vivo fibrogenic effect of ET-1 is not well characterized. We used the adenoviral-mediated gene transfer of ET-1 to overexpress ET-1 transiently in murine lungs by intratracheal administration. An increased expression of ET-1 for 3 to 10 days after injection resulted in a moderate but reversible fibrotic response, peaking on Day 14 after infection and characterized by the deposition of ECM components, myofibroblast formation, and a significant inflammatory infiltrate, mainly in the peribronchiolar/perivascular region. Adenoviral-mediated ET-1 overexpression activated focal adhesion kinase (FAK) both in vitro, using primary murine lung fibroblasts, and in vivo, intratracheally administered in the lungs of mice. The inhibition of FAK with the compound PF-562,271 prevented ET-1-mediated collagen deposition and myofibroblast formation, thereby preventing the development of lung fibrosis. In conclusion, we demonstrate that the overexpression of ET-1 directly in the lungs of mice can initiate a fibrogenic response characterized by increased ECM deposition and myofibroblast formation, and that this effect of ET-1 can be prevented by inhibition of FAK. Our data suggest that the ET-1/FAK axis may contribute importantly to the pathogenesis of fibrotic disorders, and highlight FAK as a potential therapeutic target in these devastating diseases.

The non-canonical NOTCH ligand DLK1 exhibits a novel vascular role as a strong inhibitor of angiogenesis.

AIMS: The epidermal growth factor-like protein Delta-like 1 (DLK1) regulates multiple differentiation processes. It resembles NOTCH ligands structurally and is considered a non-canonical ligand. Given the crucial role of the NOTCH pathway in angiogenesis, we hypothesized that DLK1 could regulate angiogenesis by interfering with NOTCH. We therefore investigated the expression and function of DLK1 in the vascular endothelium and its role in the regulation of angiogenesis. METHODS AND RESULTS: We report DLK1 expression in the endothelium of different species, including human, cow, pig, and mouse. Angiogenesis was studied by using in vitro and in vivo models of angiotube formation in endothelial cells, retinal phenotypes in Dlk1-null mice, and vessel development in zebrafish. DLK1 overexpression strongly inhibited angiotube formation, whereas lung endothelial cells from Dlk1-null mice were highly angiogenic. In vivo studies demonstrated DLK1-mediated inhibition of neovessel formation and revealed an altered pattern of angiogenesis in the retinas of Dlk1-null mice. The expression of human DLK1 in zebrafish embryos severely altered the formation of intersegmental vessels, while knockdown of the orthologous gene was associated with ectopic and increased tumour-induced angiogenesis. NOTCH-dependent signalling as determined by gene expression reporters was inhibited by the presence of DLK1 in vascular endothelial cells. In contrast, Dlk1-null mice showed increased levels of NOTCH downstream targets, such as Snail and Slug. CONCLUSION: Our results unveil a novel inhibitory role for DLK1 in the regulation of angiogenesis, mediated by antagonism of the NOTCH pathway, and establish the basis for investigating its action in pathological settings.