THE EMILIN1-\u3b14/\u3b19 INTEGRIN INTERACTION IS CRUCIAL IN LYMPHATIC VESSEL FORMATION AND MAINTENANCE

Lymphatic system is the second circulatory system and it is crucial for the maintenance of tissue interstitial fluid balance and it plays important roles in human diseases, including lymphedema, chronic inflammation and cancer metastasis. Lymphatic vasculature is divided essentially in two types of vessels: capillaries and collectors. Lymphatic capillaries allow absorption of fluid from interstitium whereas lymphatic collectors transport the lymph to lymph nodes and in the end in the blood circulation. A previous work demonstrated that EMILIN1, an extracellular matrix glycoprotein associated with elastic fibers, has a crucial role in the proper formation of lymphatic capillaries. Emilin1-/- mouse is the first abnormal lymphatic phenotype associated with an extracellular matrix component deficiency: enlargement of lymphatic vessels in several tissues and reduction of anchoring filaments. This morphological alterations cause impair of lymph uptake, increase of leakage and the development of a mild lymphedema condition. Moreover, through the engagement with by \u3b14\u3b21 integrin, EMILIN1 exerts a regulatory role in cell proliferation: in Emilin1-/- background there is an increase of proliferative rate. In this study we focus our interest on the role of EMILIN1 in the lymphatic vessel formation process. Our results show that EMILIN1, expressed in embryonic, postnatal and adult mouse valve structures, regulates lymphatic valve formation, maturation and maintenance. Emilin1-/- mice exhibit an immature vessel and valve leaflet structures, which affect also their functionality. We demonstrate that also in the lymphatic contest EMILIN1 is able to regulate cell proliferation. Moreover this extracellular matrix protein influences also the migratory ability of the endothelial lymphatic cells. The regulatory ability of EMILIN1 in the lymphatic is mediated by \u3b19\u3b21 integrin engagement. EMILIN1 influences the lymphangiogenesis process not only in the physiological but also in the pathological environment: it exerts a protective role in tumor growth, in tumor lymphangiogenesis as well as in metastatic spread to lymph nodes. Our data demonstrate for the first time a fundamental role for EMILIN1-\u3b19\u3b21 integrin interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance. Moreover our data underline the importance of this extracellular matrix component in displaying a regulatory function in proliferation and acting as a \u201cguiding\u201d molecule in migration of lymphatic endothelial cells

Local inhibition of elastase reduces EMILIN1 cleavage reactivating lymphatic vessel function in a mouse lymphoedema model

Lymphatic vasculature critically depends on the connections of lymphatic endothelial cells with the extracellular matrix (ECM), which are mediated by anchoring filaments (AFs). The ECM protein EMILIN1 is a component of AFs and is involved in the regulation of lymphatic vessel functions: accordingly, Emilin1(-/-) mice display lymphatic vascular morphological alterations, leading to functional defects such as mild lymphoedema, lymph leakage and compromised lymph drainage. In the present study, using a mouse post-surgical tail lymphoedema model, we show that the acute phase of acquired lymphoedema correlates with EMILIN1 degradation due to neutrophil elastase (NE) released by infiltrating neutrophils. As a consequence, the intercellular junctions of lymphatic endothelial cells are weakened and drainage to regional lymph nodes is severely affected. The local administration of sivelestat, a specific NE inhibitor, prevents EMILIN1 degradation and reduces lymphoedema, restoring a normal lymphatic functionality. The finding that, in human secondary lymphoedema samples, we also detected cleaved EMILIN1 with the typical bands of an NE-dependent pattern of fragmentation establishes a rationale for a powerful strategy that targets NE inhibition. In conclusion, the attempts to block EMILIN1 degradation locally represent the basis for a novel 'ECM' pharmacological approach to assessing new lymphoedema treatments

The mutational landscape of the adult healthy parous and nulliparous human breast

Abstract The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland

The mutational landscape of the adult healthy parous and nulliparous human breast

The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.</p

The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection

<div><p>Bacterial genotoxins, produced by several Gram-negative bacteria, induce DNA damage in the target cells. While the responses induced in the host cells have been extensively studied <i>in vitro</i>, the role of these effectors during the course of infection remains poorly characterized. To address this issue, we assessed the effects of the <i>Salmonella enterica</i> genotoxin, known as typhoid toxin, in <i>in vivo</i> models of murine infection. Immunocompetent mice were infected with isogenic <i>S</i>. <i>enterica</i>, serovar Typhimurium (<i>S</i>. Typhimurium) strains, encoding either a functional or an inactive typhoid toxin. The presence of the genotoxic subunit was detected 10 days post-infection in the liver of infected mice. Unexpectedly, its expression promoted the survival of the host, and was associated with a significant reduction of severe enteritis in the early phases of infection. Immunohistochemical and transcriptomic analysis confirmed the toxin-mediated suppression of the intestinal inflammatory response. The presence of a functional typhoid toxin further induced an increased frequency of asymptomatic carriers. Our data indicate that the typhoid toxin DNA damaging activity increases host survival and favours long-term colonization, highlighting a complex cross-talk between infection, DNA damage response and host immune response. These findings may contribute to understand why such effectors have been evolutionary conserved and horizontally transferred among Gram-negative bacteria.</p></div

Phylogenetic analysis of the intestinal microbiota.

<p>Relative abundance of the significantly altered bacterial families in the intestinal microbiota of mice infected with the MC1-TT (TT) or MC1-Δ<i>cdtB</i> (Δ<i>cdtB</i>) strain compared to uninfected mice. *p value ≤ 0.05.</p

qPCR-Array analysis of colon and liver 180 days p.i.

<p><b>A.</b> Violin plot representing the distribution of the log₂ fold changes of transcripts in the indicated tissues of mice infected with the MC1-TT strain compared to those detected in mice infected with the MC1-∆<i>cdtB</i> strain at 180 days p.i. Statistical analysis was performed with the Wilcoxon rank-sum test. Colon: p-value = 4.989e-13 (distribution of log2 fold changes is significantly shifted below 0). Liver: p-value = 0.03676 (distribution of log2 fold changes is moderately shifted over 0). ****p value ≤ 0.0001, *p ≤ 0.05. <b>B.</b> Heat map and functional annotation analysis for genes down- and up-regulated identified in A.</p

Infection with genotoxic <i>Salmonella</i> strains promotes survival of the host.

<p><b>A.</b> Percentage of survival during the infection with the virulent MC1 (left panel) or the attenuated MC71 (right panel) strain. The Kaplan-Meier method was used to evaluate survival (95% confidence interval). *p value ≤ 0.05. <b>B.</b> Dissemination of <i>S</i>. Typhimurium in cecum, mesenteric lymph nodes (MLNs), liver, and spleen in mice infected for 10 days with the control or toxigenic MC1 or MC71 strains. Data are presented as colony forming unit (CFU) per organ. Statistical analysis was performed using the Student t-test (n mice = 6).</p