INTERAZIONE COMPLEMENTO-ENDOTELIO NELLA FLOGOSI

2003/2004Numerose molecole circolanti nel sangue possono interagire con l'endotelio portando all'attivazione della cascata della coagulazione o all'insorgenza dei fenomeni proinfiammatori. Tra le molecole che possono reagire con le cellule endoteliali (EC) ci sono anche i prodotti di attivazione del sistema del complemento (C). Il sistema del C è un componente dell'immunità naturale, che svolge un ruolo essenziale nella difesa contro agenti infettivi e nella rimozione degli immunocomplessi, e rappresenta un importante mediatore del processo flogistico. L'attivazione della sequenza complementare può avvenire essenzialmente attraverso tre vie che portano prima all'attivazione del C3 e, successivamente, tramite la produzione del C3b, alla formazione delle C5 convertasi. Tale molecola scinde il C5 liberando il C5a ed il C5b, che espone il sito di legame per i componenti successivi. Quando il C5b6 si lega al C7, il complesso va incontro ad una transizione strutturale idrofilica-anfifilica per cui, per alcuni millisecondi, espone siti di legame per i fosfolipidi di membrana. Il complesso C5b67 che si forma sulla membrana di una cellula bersaglio si inserisce nel doppio strato lipidico e lega il C8 ed il C9, che polimerizza formando il MAC (Membrane Attack Complex), e causa la lisi del bersaglio. Se la reazione avviene in fase fluida, il C5b67 perde rapidamente la capacità di legarsi a membrane cellulari attraverso il sito idrofobico e diffonde nel microambiente, mantenendo la capacità di legare i successivi componenti terminali C8 e C9. Si forma così un complesso terminale citoliticamente inattivo (iTCC). Il complesso terminale che si trova in circolo, in seguito alla attivazione del sistema, viene più frequentemente definito SC5b-9, in quanto presenta anche la proteina S o vitronectina. Studi condotti nel nostro laboratorio hanno permesso di documentare che l'iTCC stimola l'espressione di ELAM, di VCAM-le di ICAM-1 sulle EC e promuove la flogosi favorendo l'adesione, la migrazione e l'accumulo tissutale di neutrofili. Il primo obiettivo di questa tesi è stato quindi quello di valutare il contributo dell'iTCC e della sua forma presente nel torrente circolatorio, l'SC5b-9, all'induzione di un'altra manifestazione della flogosi, la permeabilità vascolare. Inizialmente, abbiamo voluto accertare se il complesso potesse promuovere l'aumento della permeabilità endoteliale, utilizzando un modello "in vitro". Abbiamo poi studiato il ruolo dell'endotelio in questo fenomeno andando a considerare quali potessero essere i fattori coinvolti nell'aumento della permeabilità. Sono stati utilizzati particolari inibitori del Platelet-Activating Factor (PAF) e del sistema delle chinine, che risultano essere molecole particolarmente coinvolte nelle risposte endoteliali durante questa fase della flogosi. I dati ottenuti con il modello "in vitro" sono stati supportati anche da esperimenti condotti "in vivo" nel ratto. Infine abbiamo voluto esaminare l'effetto dell' SC5b-9 sull'integrità dell'endotelio utilizzando tecniche di microscopia elettronica a trasmissione su sezioni "ex vivo".  La formazione del complesso terminale avviene nel torrente circolatorio, e si deposita su alcuni tessuti in condizioni patologiche. E' noto, infatti, che i componenti complementari, compresi quelli terminali, sono sintetizzati non solo dalle cellule epatiche ma anche da molte altre cellule presenti nei tessuti, come macrofagi, fibroblasti e cellule endoteliali. Studi condotti nel nostro laboratorio avevano dimostrato che le HUVEC sono in grado di sintetizzare C3 e C7. Il secondo obiettivo della tesi è stato quello di esaminare la possibilità che il C7 sia presente sulla membrana delle EC ed agisca da accettore silente per i componenti complementari terminali attivati, garantendo alle EC una protezione contro un'eccessiva attivazione del sistema. Per valutare la presenza dei componenti C3 e C7 sulla membrana sono state applicate diverse tecniche comprendenti l 'ELISA, la microsopia a fluorescenza e quella elettronica a trasmissione. Inoltre, dopo aver dimostrato la presenza sulla membrana cellulare del C7 ma non del C3, abbiamo studiato i possibili ruoli funzionali di tale molecola, accertando una eventuale attività di "decoy acceptor" per gli altri componenti complementari. In particolare, si è valutato un possibile effetto protettivo sulle EC, svolto dal C7 di membrana, nei confronti del TCC solubile. Abbiamo studiato quindi come la formazione del complesso terminale sul C7 di membrana (mTCC) inibisca l'effetto proinfiammatorio svolto dal TCC solubile sulle EC, valutando l'espressione delle molecole di adesione, la produzione di IL-8 e l'aumento della permeabilità vascolare. Nella prima parte di questa tesi abbiamo dimostrato che sia l'iTCC sia il complesso presente in circolo, l'SC5b-9, inducono aumento della permeabilità vascolare, promuovendo la flogosi. L'attività permeabilizzante svolta dal TCC è mediata dal rilascio da parte delle EC di molecole vasoattive quali PAF e bradi chinina (BK). I risultati ottenuti utilizzando modelli "in vitro", sono stati confermati anche in un modello "in vivo" nel ratto. L' SCSb-9 induce la permeabilità vascolare provocando l'apertura delle giunzioni intercellulari. Abbiamo dimostrato, inoltre, che l'effetto vasopermeabilizzante provocato dal complesso terminale viene inibito dagli antagonisti dei recettori della BK di tipo B2 e del PAF. Nella seconda parte abbiamo valutato l'espressione dei componenti del C sulla membrana delle EC. Abbiamo dimostrato che il C7 è presente sulle EC isolate da vari distretti tissutali, e che è covalentemente legato alla membrana. L'espressione del C7 è modulata da diverse citochine e la molecola, simile a quella solubile, mantiene la capacità di legare gli altri componenti terminali formando il TCC sulla membrana cellulare (mTCC). Il C7 di membrana svolge una funzione di "decoy acceptor" per il TCC nascente, ed è in grado di inibire tutte le attività proinfiammatorie del complesso solubile, quali l'espressione delle molecole di adesione, la secrezione della IL-8 e l'aumento della permeabilità vascolare.XVII Ciclo1975Versione digitalizzata della tesi di dottorato cartacea

Roles and Clinical Applications of OPG and TRAIL as Biomarkers in Cardiovascular Disease

Cardiovascular diseases (CVD) remain the major cause of death and premature disability in Western societies. Assessing the risk of CVD is an important aspect in clinical decision-making. Among the growing number of molecules that are studied for their potential utility as CVD biomarkers, a lot of attention has been focused on osteoprotegerin (OPG) and its ligands, which are receptor activator of nuclear factor \u3baB ligand (RANKL) and TNF-related apoptosis-inducing ligand. Based on the existing literature and on our experience in this field, here we review what the possible roles of OPG and TRAIL in CVD are and their potential utility as CVD biomarkers

Platelet-Activating Factor and Kinin-Dependent Vascular Leakage as a Novel Functional Activity of the Soluble Terminal Complement Complex

AbstractThe infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy

RelB activation in anti-inflammatory decidual endothelial cells: a master plan to avoid pregnancy failure?

It is known that excessive inflammation at fetal-maternal interface is a key contributor in a compromised pregnancy. Female genital tract is constantly in contact with microorganisms and several strategies must be adopted to avoid pregnancy failure. Decidual endothelial cells (DECs) lining decidual microvascular vessels are the first cells that interact with pro-inflammatory stimuli released into the environment by microorganisms derived from gestational tissues or systemic circulation. Here, we show that DECs are hypo-responsive to LPS stimulation in terms of IL-6, CXCL8 and CCL2 production. Our results demonstrate that DECs express low levels of TLR4 and are characterized by a strong constitutive activation of the non-canonical NF-\u3baB pathway and a low responsiveness of the canonical pathway to LPS. In conclusion, DECs show a unique hypo-responsive phenotype to the pro-inflammatory stimulus LPS in order to control the inflammatory response at feto-maternal interface

the neutrophil activating protein of helicobacter pylori crosses endothelia to promote neutrophil adhesion in vivo

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of β2 integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection

MBL Interferes with Endovascular Trophoblast Invasion in Pre-Eclampsia

The spiral arteries undergo physiologic changes during pregnancy, and the failure of this process may lead to a spectrum of pregnancy disorders, including pre-eclampsia. Our recent data indicate that decidual endothelial cells (DECs), covering the inner side of the spiral arteries, acquire the ability to synthesize C1q, which acts as a link between endovascular trophoblast and DECs favouring the process of vascular remodelling. In this study, we have shown that sera obtained from pre-eclamptic patients strongly inhibit the interaction between extravillous trophoblast (EVT) and DECs, preventing endovascular invasion of trophoblast cells. We further demonstrated that mannose-binding lectin (MBL), one of the factor increased in pre-eclamptic patient sera, strongly inhibits the interaction of EVT with C1q interfering with the process of EVT adhesion to and migration through DECs. These data suggest that the increased level of MBL in pre-eclampsia may contribute to the failure of the endovascular invasion of trophoblast cells

A transcriptomic study of Hereditary Angioedema attacks

Background: Hereditary angioedema (HAE) caused by C1-inhibitor deficiency is a lifelong illness characterized by recurrent acute attacks of localized skin or mucosal edema. Activation of the kallikrein/bradykinin pathway at the endothelial cell level has a relevant pathogenetic role in acute HAE attacks. Moreover, other pathways are involved given the variable clinical expression of the disease in different patients. Objective: We sought to explore the involvement of other putative genes in edema formation. Methods: We performed a PBMC microarray gene expression analysis on RNA isolated from patients with HAE during an acute attack and compared them with the transcriptomic profile of the same patients in the remission phase. Results: Gene expression analysis identified 23 genes significantly modulated during acute attacks that are involved primarily in the natural killer cell signaling and leukocyte extravasation signaling pathways. Gene set enrichment analysis showed a significant activation of relevant biological processes, such as response to external stimuli and protein processing (q < 0.05), suggesting involvement of PBMCs during acute HAE attacks. Upregulation of 2 genes, those encoding adrenomedullin and cellular receptor for urokinase plasminogen activator (uPAR), which occurs during an acute attack, was confirmed in PBMCs of 20 additional patients with HAE by using real-time PCR. Finally, in vitro studies demonstrated the involvement of uPAR in the generation of bradykinin and endothelial leakage. Conclusions: Our study demonstrates the increase in levels of adrenomedullin and uPAR in PBMCs during an acute HAE attack. Activation of these genes usually involved in regulation of vascular tone and in inflammatory response might have a pathogenic role by amplifying bradykinin production and edema formation in patients with HAE

Endothelial cells and the complement component C1q as novel therapeutic tools for the treatment of chronic ulcers

One of the major limitation of the current treatments for burns and chronic ulcers is the absence of a rapid functional vascular plexus formation. The impaired angiogenic condition leads to a poor nutritional intake and to increased microbial contamination. To evaluate novel potential therapeutic strategies to improve new vessel formation we set up a method for the isolation of human adult dermal endothelial cells (ADMEC) from skin biopsies. We then evaluated the ability of ADMEC to adhere and to grow into a tridimensional matrix of collagen and of human decellularized dermis. A wound healing model was established in rats to investigate the role of endothelial cells in an in vivo angiogenic process. The data show an increase of vascular structures in the wounds treated with endothelial cells compared to the controls. Several soluble factors can be produced by endothelial cells and promote angiogenesis. We have recently showed that decidual endothelial cells are able to synthesize C1q and express surface-bound C1q under physiological conditions. Since decidua is a site of active angiogenesis, we sought to ascertain whether C1q can play a role in this process. To confirm our hypothesis we used different approaches such as permeability, cell migration and proliferation assay, besides wound healing and aortic ring assay. C1q acts as a permeabilizing factor inducing the FITC-BSA leakage through a monolayer of endothelial cells (ECs). Next, we found that C1q was able to promote motility of ECs in a wound healing assay, and to recruit ECs acting as a chemotactic factor, furthermore C1q was also found to have an additional effect on EC inducing cell proliferation. To confirm and extend these data, we used the rat aortic ring assay to evaluate the ex vivo effect of C1q. C1q was also found to stimulate the formation of tubular structures in a matrigel assay and to promote sprouting formation in the aortic ring assay. The presence of both ECs and pericytes were documented in the sprouts indicating that complete new vessels are being formed. The in vivo proangiogenic activity of C1q was evaluated in rats using a wound healing assay. C1q, VEGF or saline was topically applied to the wounds and the skin lesions removed after 14 days were examined for vessel formation. The wounds treated with C1q exhibited a number of new vessels increased to that of saline treated wounds and comparable to that of VEGF. The results suggest that the topical application of endothelial cells or C1q are of potential therapeutic interest as a pro angiogenic treatment of chronic ulcers

TRAIL reduces impaired glucose tolerance and NAFLD in the high-fat diet-fed mouse

Recent studies suggest that a circulating protein called TRAIL (TNF-related apoptosis inducing ligand) may have an important role in the treatment of type 2 diabetes. It has been shown that TRAIL deficiency worsens diabetes and that TRAIL delivery, when it is given before disease onset, slows down its development. The present study aimed at evaluating whether TRAIL had the potential not only to prevent, but also to treat type 2 diabetes. Thirty male C57BL/6J mice were randomized to a standard or a high-fat diet (HFD). After 4 weeks of HFD, mice were further randomized to receive either placebo or TRAIL, which was delivered weekly for 8 weeks. Body weight, food intake, fasting glucose, and insulin were measured at baseline and every 4 weeks. Tolerance tests were performed before drug randomization and at the end of the study. Tissues were collected for further analyses. Parallel in vitro studies were conducted on HepG2 cells and mouse primary hepatocytes. TRAIL significantly reduced body weight, adipocyte hypertrophy, free fatty acid levels, and inflammation. Moreover, it significantly improved impaired glucose tolerance, and ameliorated non-alcoholic fatty liver disease (NAFLD). TRAIL treatment reduced liver fat content by 47% in vivo as well as by 45% in HepG2 cells and by 39% in primary hepatocytes. This was associated with a significant increase in liver peroxisome proliferator-activated receptor (PPAR) \u3b3 (PPAR\u3b3) co-activator-1 \u3b1 (PGC-1\u3b1) expression both in vivo and in vitro, pointing to a direct protective effect of TRAIL on the liver. The present study confirms the ability of TRAIL to significantly attenuate diet-induced metabolic abnormalities, and it shows for the first time that TRAIL is effective also when administered after disease onset. In addition, our data shed light on TRAIL therapeutic potential not only against impaired glucose tolerance, but also against NAFLD