Targeting interleukin-1β protects from aortic aneurysms induced by disrupted transforming growth factor β signaling

Aortic aneurysms are life-threatening conditions with effective treatments mainly limited to emergency surgery or trans-arterial endovascular stent grafts, thus calling for the identification of specific molecular targets. Genetic studies have highlighted controversial roles of transforming growth factor β (TGF-β) signaling in aneurysm development. Here, we report on aneurysms developing in adult mice after smooth muscle cell (SMC)-specific inactivation of Smad4, an intracellular transducer of TGF-β. The results revealed that Smad4 inhibition activated interleukin-1β (IL-1β) in SMCs. This danger signal later recruited innate immunity in the adventitia through chemokine (C-C motif) ligand 2 (CCL2) and modified the mechanical properties of the aortic wall, thus favoring vessel dilation. SMC-specific Smad4 deletion in Il1r1- or Ccr2-null mice resulted in milder aortic pathology. A chronic treatment with anti-IL-1β antibody effectively hampered aneurysm development. These findings identify a mechanistic target for controlling the progression of aneurysms with compromised TGF-β signaling, such as those driven by SMAD4 mutations

Serious Delayed Hair Toe Tourniquet Syndrome with Bone Erosion and Flexor Tendon Lesion

Hair toe tourniquet syndrome (HTTS) is an uncommon pediatric condition occurring when the toe is circumferentially strangulated by human hair or fibers. An 8-week-old little girl was admitted to the Emergency Department because of the worsening swelling in the right second and third toes, which had been been previously treated with a local antibiotic thinking of an infection. An unrecognized HTTS was leading the third toe to necrosis. An urgent release of the constricting band on the two toes was performed and bone erosion and partial flexor tendon lesion on the third toe were detected. We would like to raise awareness in the community and in colleagues about HTTS in children, because early recognition and urgent treatment are mandatory to provide an adequate management and prevent severe complications

Electrospun Structures Made of a Hydrolyzed Keratin-Based Biomaterial for Development of in vitro Tissue Models

The aim of this study is the analysis and characterization of a hydrolyzed keratin-based biomaterial and its processing using electrospinning technology to develop in vitro tissue models. This biomaterial, extracted from poultry feathers, was mixed with type A porcine gelatin and cross-linked with γ-glycidyloxy-propyl-trimethoxy-silane (GPTMS) to be casted initially in the form of film and characterized in terms of swelling, contact angle, mechanical properties, and surface charge density. After these chemical-physical characterizations, electrospun nanofibers structures were manufactured and their mechanical properties were evaluated. Finally, cell response was analyzed by testing the efficacy of keratin-based structures in sustaining cell vitality and proliferation over 4 days of human epithelial, rat neuronal and human primary skin fibroblast cells

multimerin 2 maintains vascular stability and permeability

Abstract Multimerin-2 is an extracellular matrix glycoprotein and member of the elastin microfibril interface-located (EMILIN) family of proteins. Multimerin-2 is deposited along blood vessels and we previously demonstrated that it regulates the VEGFA/VEGFR2 signaling axis and angiogenesis. However, its role in modulating vascular homeostasis remains largely unexplored. Here we identified Multimerin-2 as a key molecule required to maintain vascular stability. RNAi knockdown of Multimerin-2 in endothelial cells led to cell-cell junctional instability and increased permeability. Mechanistically cell-cell junction dismantlement occurred through the phosphorylation of VEGFR2 at Tyr951, activation of Src and phosphorylation of VE-cadherin. To provide an in vivo validation for these in vitro effects, we generated Multimerin-2−/− (Mmrn2−/−) mice. Although Mmrn2−/− mice developed normally and displayed no gross abnormalities, endothelial cells displayed cell junctional defects associated with increased levels of VEGFR2 phospho-Tyr949 (the murine counterpart of human Tyr951), impaired pericyte recruitment and increased vascular leakage. Of note, tumor associated vessels were defective in Mmrn2−/− mice, with increased number of small and often collapsed vessels, concurrent with a significant depletion of pericytic coverage. Consequently, the Mmrn2−/− vessels were less perfused and leakier, leading to increased tumor hypoxia. Chemotherapy efficacy was markedly impaired in Mmrn2−/− mice and this was associated with poor drug delivery to the tumor xenografts. Collectively, our findings demonstrate that Multimerin-2 is required for proper vessel homeostasis and stabilization, and unveil the possibility to utilize expression levels of this glycoprotein in predicting chemotherapy efficacy

Emilin-1 controls arterial blood pressure by regulating contractility of vascular smooth muscle cells

Emilin-1 is a protein of the elastic extracellular matrix (ECM) expressed in interstitial connective tissue and in the cardiovascular system. Emilin1 null mice display hypotrophic remodeling of the wall of conductance arteries and increased blood pressure. The protein regulates the bioavailability of TGF-b by inhibiting proteolysis of the proTGF-b precursor to LAP/TGF-b, a complex from which the growth factor can be subsequently released for receptor binding. In the absence of Emilin-1, the amount of active TGF-b is increased. As Emilin-1 is expressed in blood vessels starting from early stages of embryonic development to adulthood, a key question concerning the function of the protein is whether the Emilin1-/- phenotype is the result of a developmental defect or the function of the protein is required for the regulation of blood pressure and arterial structure also in the adult. The conditional gene targeting procedure chosen to inactivate the Emilin1 gene in smooth muscle cells (SMCs) of adult mice included the use of floxed Emilin1 and CreERT2 (a tamoxifen inducible Cre recombinase) under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. Tamoxifen administration induced activity of Cre specifically in vascular and visceral SMCs, as revealed by X-gal staining of tissues from animals with the Rosa26R mutation. When Emilin1flox/flox mice carrying the Smmhc-CreERT2 transgene were given tamoxifen for 7 days, Emilin-1 disappeared completely in 10-12 days from start of treatment. In the same time, blood pressure increased of about 20 mmHg, a level that was stably maintained thereafter. The myogenic response of second branch meseteric arteries, evaluated using a pressure myograph, was found to be increased in Emilin1-/- mice. Additional experiments with aorta and mesenteric artery SMC cultures from control and mutant mice showed that lack of Emilin-1  enhanced phosphorylation of myosin light chain 20 when cells were stimulated with the a1-adrenergic receptor agonists phenylephrine or with angiotensin II. Moreover, basal cytosolic Ca2+ levels and calcium transients induced by stimulation with phenylephrine and angiotensin II were increased in SMCs from Emilin1-/- mutants. The data suggest that Emilin-1 expression is continuously required for regulation of blood pressure and that the increase of TGF-b activity induced by diminished Emilin-1  stimulates, likely through alteration of intracellular calcium homeostasis, contractility of vascular SMC to mechanical and chemical stimuli with ensuing hypertension

Involvement of TGF-?1 in Multimerin-2, but not Emilin-2 regulation of blood pressure

Emilins are a family of extracellular matrix (ECM) glycoproteins characterized by a cysteine-rich N-terminal EMI domain. The family comprises four members in mammals: Emilin-1, Emilin-2, Multimerin-1 and Multimerin-2. The prototype of this family is Emilin-1, a protein widely distributed in interstitial connective tissue in association with elastic fibers and strongly expressed in the mouse cardiovascular system during development and in the adult. Emilin1 knockout animals display increased blood pressure, increased peripheral vascular resistance, and reduced size of arterial tunica media. The mechanism that brings about this phenotype entails an increase of TGF-?1 signalling with consequences on vascular SMC growth, ECM homeostasis, and vascular remodelling. It has been found that Emilin-1, through its EMI domain, binds proTGF-?1 and prevents its maturation by proprotein convertases. Therefore, Emilin-1 has an important role in the regulation of TGF-? extracellular availability. To gain insight into the function of two other members of the Emilin family, Emilin2 and Mmrn2, knockout mice for these genes were generated. Both genes are expressed mainly in the cardiovascular system: during development Emilin-2 was mainly found in the heart and blood vessels of the central nervous system, while in the adult it was detected in lymphoid organs and, with fainter staining, in heart and kidney. The expression of Multimerin-2 was restricted to endothelium. The pattern of expression of these genes and the high sequence similarity with Emilin-1 (particularly in EMI domain) stimulated the analysis of the phenotype of the cardiovascular system in mutant mice. Mmrn2 and Emilin2 knockout mice were found to be hypertensive. For Multimerin-2 this alteration is accompanied by a mild reduction of the media cross-sectional area, whereas this is not the case for Emilin-2. Moreover, Mmrn2 null mice exhibit an increased contraction of resistance vessels in response to the adrenergic ?1 agonist phenylephrine. On the contrary, the response of Emilin2-/- blood vessels to this drug was normal. Molecular mechanisms by witch Emilin-2 and Multimerin-2 carry out their physiological function in blood vessels were studied through in vivo and in vitro biochemical study. Evidences in vitro demonstrate that Emilin-2 and Multimerin-2 could reduce TGF-?1 signalling through the inhibition of proTGF-?1 processing into the LAP/TGF-? complex. This activity is mediated by the EMI domain and the formation of a supramolecular association of Emilins with proTGF-?1 (or the LAP/TGF-? complex) was demonstrated. The relevance of TGF-?1 in the pathogenesis of cardiovascular phenotype was tested by experiments in vivo, in which the TGF-?1 gene dosage was genetically reduced in Emilin2 and Mmrn2 null mice. The hypertensive phenotype was rescued in Mmrn2 knockout mice, while the blood pressure remained elevated in Emilin2 null animals. Different isoforms of TGF-? are expressed in the cardiovascular system during development and the inhibitory effect of Emilin-2 on TGF-?2 and -?3 signalling was demonstrated through in vitro assays. However, inactivation of a single TGF-?2 allele did not reverse the hypertensive phenotype of Emilin2-/- mice, indicating that TGF-?2 dysregulation is not involved in the pathogenesis of this phenotype. In conclusion the observations that Multimerin-2 produced by endothelial cells is able to regulate contractility of arterial vessels (due to vascular SMC) to adrenergic stimulation and that the hypertensive phenotype of null mice is caused by increased TGF-?1 signalling led to hypothesize that endothelial cells through expression of TGF-?1 regulate contractility of vascular SMC. Multimerin-2, localized between endothelial cells and vascular SMC, reduces the amount of TGF-?1 available to vascular SMC. Therefore, lack of Multimerin-2 gives rise to an increased signalling of TGF-?1 in vascular SMC that became more susceptible to sympathetic stimulation. Increased contractility of vascular SMC in small resistance vessels increases peripheral resistance and leads to hypertension. The cytostatic effect of TGF-?1 on vascular SMC and the consequent reduction in vessels size contribute to the generation of hypertensive phenotype. The mechanism of regulation of blood pressure by Emilin-2 remains to be elucidated. The role of TGF-?3 in blood pressure homeostasis supposed from data in vitro needs to be verified in vivo. The involvement of other members of the TGF-? family of growth factors will also be considered in the future.Le Emiline costituiscono una famiglia di glicoproteine della matrice extracellulare caratterizzate dalla presenza, all’estremità amino-terminale, di un dominio ricco di cisteine, denominato EMI. Nei mammiferi la famiglia comprende 4 membri: Emilina-1, Emilina-2, Multimerina-1, Multimerina-2. Emilina-1 è il prototipo per questa famiglia ed è largamente distribuita, in associazione con le fibre elastiche, nei tessuti connettivi interstiziali. Il sito di maggior espressione di Emilina-1 è il sistema cardiovascolare, sia durante lo sviluppo embrionale, sia nell’adulto. Animali deficienti di Emilina-1 presentano una elevata pressione sanguigna associata ad un aumento delle resistenze vascolari periferiche ed una riduzione del diametro della tonaca media delle arterie. Il meccanismo che porta a questo fenotipo coinvolge un aumento di segnalazione di TGF-?1 con effetti riguardanti la crescita delle cellule muscolari lisce vascolari, l’omeostasi della matrice extracellulare e il rimodellamento vascolare. E’ stato dimostrato che Emilina-1, mediante il dominio EMI, lega il proTGF-?1 e previene la sua maturazione da parte di proprotein convertasi. Emilina-1 ha quindi un importante ruolo nella regolazione della disponibilità del TGF-? nell’ambiente extracellulare. Per comprendere la funzione di Emilina2 e Multimerina2 sono stati generati topi con un’inattivazione genica mirata di questi geni. Il sito di maggiore espressione di entrambi i geni è il sistema cardiovascolare. Durante lo sviluppo embrionale, Emilina-2 è principalmente presente nel cuore e nei vasi del sistema nervoso centrale mentre nell’adulto è stata trovata negli organi linfoidi e, in piccola quantità, nel cuore e nel rene. L’espressione di Multimerina-2 è invece ristretta agli endoteli. La distribuzione di queste proteine e la loro elevata omologia di sequenza con Emilina-1 (in particolare a livello del dominio EMI) hanno guidato l’analisi del fenotipo dei topi mutanti verso il sistema cardiovascolare. I topi deficienti di Emilina-2 e Multimerina-2 sono ipertesi. Per quanto riguarda Multimerina-2, il fenotipo ipertensivo è accompagnato da una leggera riduzione dell’area della tonaca media in sezione trasversale. Inoltre i topi deficienti di Multimerina-2 presentano un’aumentata contrattilità dei vasi di resistenza in risposta alla fenilefrina, un agonista dei recettori ?1 adrenergici. Nei topi deficienti di Emilina-2 invece questi due parametri risultano essere paragonabili ai topi di ceppo selvatico. I meccanismi molecolari attraverso i quali Emilina-2 e Multimerina-2 svolgono le loro funzioni fisiologiche nei vasi sanguigni sono stati studiati attraverso studi in vivo e studi biochimici in vitro. Questi ultimi hanno dimostrato che Emilina-2 e Multimerina-2 possono ridurre la segnalazione di TGF-?1 attraverso l’inibizione della trasformazione del proTGF-?1 nel complesso LAP/TGF-?. Questa attività, mediata dal dominio EMI, si sviluppa attraverso la formazione di un’associazione sovramolecolare con il proTGF-? (o il complesso LAP/TGF-?). L’importanza del TGF-?1 nella patogenesi del fenotipo cardiovascolare è stata testata attraverso esperimenti in vivo. Il dosaggio genico di TGF-?1 è stato geneticamente ridotto nei topi deficienti di Emilina-2 e Multimerin-2. Il fenotipo ipertensivo è stato perso dai topi con inattivazione del gene Multimerina2 mentre la pressione sanguigna rimane elevata nei topi in cui il gene Emilina2 è stato inattivato. Durante lo sviluppo embrionale, le diverse isoforme di TGF-? (TGF-?1, -?2 e -?3) sono espresse nel sistema cardiovascolare. Mediante saggi in vitro è stato dimostrato che Emilina-2 ha un effetto inibitorio anche sulla segnalazione di TGF-?2 e TGF-?3. Tuttavia, l’inattivazione di un allele di TGF-?2 in topi deficienti di Emilina-2 non porta alla perdita del fenotipo, indicando che una deregolazione della segnalazione di TGF-?2 non è coinvolta nella patogenesi di questo fenotipo. In conclusione, l’osservazione che Multimerina-2 prodotta dalle cellule endoteliali è in grado di regolare la contrazione delle arterie (dovuta alle cellule muscolari lisce vasali) in risposta alla stimolazione adrenergica e che il fenotipo ipertensivo è causato da un aumento della segnalazione di TGF-?1, porta ad ipotizzare che le cellule endoteliali, attraverso l’espressione di TGF-?1, possono regolare la contrattilità delle cellule muscolari lisce vasali. Multimerina-2, localizzata tra le cellule endoteliali e le cellule muscolari lisce, riduce la quantità di TGF-?1 disponibile per le cellule muscolari lisce. In conseguenza la mancanza di Multimerina-2 porta ad un aumento della segnalazione di TGF-?1 alle cellule muscolari lisce che diventano più sensibili alla stimolazione simpatica. Un aumento della contrattilità delle cellule muscolari lisce nei piccoli vasi di resistenza genera un aumento delle resistenze periferiche che portano quindi all’ipertensione. L’effetto citostatico del TGF-?1 sulle cellule muscolari lisce e la conseguente riduzione del calibro vasale contribuiscono alla generazione del fenotipo ipertensivo. Il meccanismo di regolazione della pressione sanguigna da parte di Emilina-2 rimane inspiegato. I dati in vitro lasciano supporre un ruolo del TGF-?3 nella regolazione della pressione sanguigna che deve però essere verificato in vivo. In futuro, inoltre, dovrà essere considerato anche il coinvolgimento di altri membri della famiglia dei TGF-?

Delayed Bleeding and Pelvic Haematoma after Low-Energy Osteoporotic Pubic Rami Fracture in a Warfarin Patient: An Unusual Cause of Abdominal Pain

Introduction. Acute abdominal pain may be the presenting symptom in a wide range of diseases in the elderly. Acute abdominal pain related to a delayed bleeding and pelvic haematoma after a low-energy pubic rami fracture is rare and can have important consequences; to the best of our knowledge, only one case has been previously described. Case Report. We present an unusual case of an 83-year-old woman taking warfarin for atrial fibrillation, admitted to the Emergency Department (ED) with acute abdominal pain and progressive anemia related to a delayed bleeding and pelvic haematoma 72 hours after a low-energy osteoporotic pubic rami fracture. Warfarin was withheld, anticoagulation was reversed by using fresh frozen plasma and vitamin K, and concentrated red blood cells were given. Haemoglobin level gradually returned to normal with a progressive resorption of the haematoma. Conclusion. Delayed bleeding and pelvic haematoma after osteoporotic pubic rami fracture should be considered in the differential diagnosis of acute abdominal pain in the elderly. This case indicates the need for hospital admission, careful haemodynamic monitoring, and early identification of bleeding in patients with “benign” osteoporotic pubic rami fracture, especially those receiving anticoagulants, to provide an adequate management and prevent severe complications