Using changes in tissue [Na + ] concentration alone as done with Na + MRI may not accurately quantitate excess tissue Na + , particularly in cellular tissues. However, individually quantitating alterations in tissue Na + and water content as possible with ashing studies may still accurately quantitate excess tissue Na + in these situations. Furthermore, when tissue [Na + ] exceeds plasma [Na + ], excess tissue Na + must be present.

Ectodomain shedding of extracellular and membrane proteins is of fundamental importance for cell–cell communication in neoplasias. A Disintegrin And Metalloproteinase (ADAM) proteases constitute a family of multifunctional, membrane-bound proteins with traditional sheddase functions. Their protumorigenic potential has been attributed to both, essential (ADAM10 and ADAM17) and ‘dispensable’ ADAM proteases (ADAM8, 9, 12, 15, and 19). Of specific interest in this review is the ADAM proteinase ADAM8 that has been identified as a significant player in aggressive malignancies including breast, pancreatic, and brain cancer. High expression levels of ADAM8 are associated with invasiveness and predict a poor patient outcome, indicating a prognostic and diagnostic potential of ADAM8. Current knowledge of substrates and interaction partners gave rise to the hypothesis that ADAM8 dysregulation affects diverse processes in tumor biology, attributable to different functional cores of the multidomain enzyme. Proteolytic degradation of extracellular matrix (ECM) components, cleavage of cell surface proteins, and subsequent release of soluble ectodomains promote cancer progression via induction of angiogenesis and metastasis. Moreover, there is increasing evidence for significance of a non-proteolytic function of ADAM8. With the disintegrin (DIS) domain ADAM8 binds integrins such as β1 integrin, thereby activating integrin signaling pathways. The cytoplasmic domain is critical for that activation and involves focal adhesion kinase (FAK), extracellular regulated kinase (ERK1/2), and protein kinase B (AKT/PKB) signaling, further contributing to cancer progression and mediating chemoresistance against first-line therapies. This review highlights the remarkable effects of ADAM8 in tumor biology, concluding that pharmacological inhibition of ADAM8 represents a promising therapeutic approach not only for monotherapy, but also for combinatorial therapies.

Abdominal aortic aneurysm (AAA) is a common age-related vascular disease characterized by progressive weakening and dilatation of the aortic wall. Thrombospondin-1 (TSP-1; gene Thbs1) is a member of the matricellular protein family important in the control of extracellular matrix (ECM) remodelling. In the present study, the association of serum TSP-1 concentration with AAA progression was assessed in 276 men that underwent repeated ultrasound for a median 5.5 years. AAA growth was negatively correlated with serum TSP-1 concentration ( Spearman’s rho − 0.129, P =0.033). Men with TSP-1 in the highest quartile had a reduced likelihood of AAA growth greater than median during follow-up (OR: 0.40; 95% confidence interval (CI): 0.19–0.84, P =0.016, adjusted for other risk factors). Immunohistochemical staining for TSP-1 was reduced in AAA body tissues compared with the relatively normal AAA neck. To further assess the role of TSP-1 in AAA initiation and progression, combined TSP-1 and apolipoprotein deficient ( Thbs1 −/− ApoE −/− , n =20) and control mice ( ApoE −/− , n =20) were infused subcutaneously with angiotensin II (AngII) for 28 days. Following AngII infusion, Thbs1 −/− ApoE −/− mice had larger AAAs by ultrasound ( P =0.024) and ex vivo morphometry measurement ( P =0.006). The Thbs1 −/− ApoE −/− mice also showed increased elastin filament degradation along with elevated systemic levels and aortic expression of matrix metalloproteinase (MMP)-9. Suprarenal aortic segments and vascular smooth muscle cells (VSMCs) isolated from Thbs1 −/− ApoE −/− mice showed reduced collagen 3A1 gene expression. Furthermore, Thbs1 −/− ApoE −/− mice had reduced aortic expression of low-density lipoprotein (LDL) receptor-related protein 1. Collectively, findings from the present study suggest that TSP-1 deficiency promotes maladaptive remodelling of the ECM leading to accelerated AAA progression.

In patients with non-alcoholic fatty liver disease (NAFLD), insulin resistance (IR) associates with fibrosis progression independently of the hepatic inflammation, but the mechanisms are still unclear. We modeled the independent contribution of inflammation (non-alcoholic steatohepatitis: NASH) by exploiting the methionine-choline deficient (MCD) diet, and that of IR by insulin receptor (InsR) haploinsufficieny (InsR+/–) in the pathogenesis of liver fibrosis in C57BL/6 mice. We confirmed the study findings in 96 patients with NAFLD. InsR+/– enhanced hepatic fat content and impaired hepatic insulin signaling leading to Forkhead box protein O1 (FoxO1) accumulation in MCD-fed mice. Remarkably, despite reduced inflammation and hampered transdifferentiation of hepatic stellate cells (HSCs), InsR+/– promoted hepatic fibrosis accumulation, which correlated with the induction of the Lysyl Oxidase Like 2 (Loxl2), involved in matrix stabilization. Loxl2 up-regulation was not a cell autonomous property of insulin resistant HSCs, but was dependent on microparticles (MPs) released specifically by insulin resistant hepatocytes (HEPs) exposed to fatty acids. The mechanism entailed FoxO1 up-regulation, as FoxO1 silencing normalized Loxl2 expression reversing fibrosis in InsR+/– MCD-fed mice. Loxl2 up-regulation was similarly detected during IR induced by obesity, but not by lipogenic stimuli (fructose feeding). Most importantly, LOXL2 up-regulation was observed in NAFLD patients with type 2 diabetes (T2D) and LOXL2 hepatic and circulating levels correlated with histological fibrosis progression. IR favors fibrosis deposition independently of the classic ‘inflammation – HSC transdifferentiation’ pathway. The mechanism entails a cross-talk between enhanced lipotoxicity in insulin resistant HEPs and Loxl2 production by HSCs, which was confirmed in patients with diabetes, thereby facilitating extracellular matrix (ECM) stabilization.

Cerebral small vessel disease (SVD) is a common cause of lacunar strokes, vascular cognitive impairment (VCI) and vascular dementia. SVD is thought to result in reduced cerebral blood flow, impaired cerebral autoregulation and increased blood–brain barrier (BBB) permeability. However, the molecular mechanisms underlying SVD are incompletely understood. Recent studies in monogenic forms of SVD, such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and ‘sporadic’ SVD have shed light on possible disease mechanisms in SVD. Proteomic and biochemical studies in post-mortem monogenic SVD patients, as well as in animal models of monogenic disease have suggested that disease pathways are shared between different types of monogenic disease, often involving the impairment of extracellular matrix (ECM) function. In addition, genetic studies in ‘sporadic’ SVD have also shown that the disease is highly heritable, particularly among young-onset stroke patients, and that common variants in monogenic disease genes may contribute to disease processes in some SVD subtypes. Genetic studies in sporadic lacunar stroke patients have also suggested distinct genetic mechanisms between subtypes of SVD. Genome-wide association studies (GWAS) have also shed light on other potential disease mechanisms that may be shared with other diseases involving the white matter, or with pathways implicated in monogenic disease. This review brings together recent data from studies in monogenic SVD and genetic studies in ‘sporadic’ SVD. It aims to show how these provide new insights into the pathogenesis of SVD, and highlights the possible convergence of disease mechanisms in monogenic and sporadic SVD.

Vascular cognitive impairment and dementia (VCID) include a wide spectrum of chronic manifestations of vascular disease related to large vessel strokes and small vessel disease (SVD). Lacunar strokes and white matter (WM) injury are consequences of SVD. The main vascular risk factor for SVD is brain hypoperfusion from cerebral blood vessel narrowing due to chronic hypertension. The hypoperfusion leads to activation and degeneration of astrocytes with the resulting fibrosis of the extracellular matrix (ECM). Elasticity is lost in fibrotic cerebral vessels, reducing the response of stiffened blood vessels in times of increased metabolic need. Intermittent hypoxia/ischaemia activates a molecular injury cascade, producing an incomplete infarction that is most damaging to the deep WM, which is a watershed region for cerebral blood flow. Neuroinflammation caused by hypoxia activates microglia/macrophages to release proteases and free radicals that perpetuate the damage over time to molecules in the ECM and the neurovascular unit (NVU). Matrix metalloproteinases (MMPs) secreted in an attempt to remodel the blood vessel wall have the undesired consequences of opening the blood–brain barrier (BBB) and attacking myelinated fibres. This dual effect of the MMPs causes vasogenic oedema in WM and vascular demyelination, which are the hallmarks of the subcortical ischaemic vascular disease (SIVD), which is the SVD form of VCID also called Binswanger's disease (BD). Unravelling the complex pathophysiology of the WM injury-related inflammation in the small vessel form of VCID could lead to novel therapeutic strategies to reduce damage to the ECM, preventing the progressive damage to the WM.

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.

In Crohn's disease, one of the two major forms of inflammatory bowel diseases in human beings, persistent and chronic inflammation promotes fibrotic processes thereby facilitating formation of strictures, the most common indication for surgical intervention in this disorder. The pathogenesis of Crohn's disease-associated fibrosis is not fully understood, but variants of genes involved in the recognition of microbial components/products [e.g. CARD15 (caspase-activating recruitment domain 15) and ATG16L1 (autophagy-related 16-like 1)] are associated with this phenotype, and experimental evidence suggests that intestinal fibrosis results from an altered balance between deposition of ECM (extracellular matrix) and degradation of ECM by proteases. Studies have also contributed to identify the main phenotypic and functional alterations of cells involved in the fibrogenic process, as well as molecules that stimulate such cells to produce elevated amounts of collagen and other ECM-related proteins. In the present review, we assess the current knowledge about cellular and molecular mediators of intestinal fibrosis and describe results of recent studies aimed at testing the preventive/therapeutic effect of compounds in experimental models of intestinal fibrosis.

VSMCs (vascular smooth muscle cells) play critical roles in arterial remodelling with aging, hypertension and atherosclerosis. VSMCs exist in diverse phenotypes and exhibit phenotypic plasticity, e.g. changing from a quiescent/contractile phenotype to an active myofibroblast-like, often called ‘synthetic’, phenotype. Synthetic VSMCs are able to proliferate, migrate and secrete ECM (extracellular matrix) proteinases and ECM proteins. In addition, they produce pro-inflammatory molecules, providing an inflammatory microenvironment for leucocyte penetration, accumulation and activation. The aging VSMCs have also shown changes in cellular phenotype, responsiveness to contracting and relaxing mediators, replicating potential, matrix synthesis, inflammatory mediators and intracellular signalling. VSMC dysfunction plays a key role in age-associated vascular remodelling. Cyclic nucleotide PDEs (phosphodiesterases), by catalysing cyclic nucleotide hydrolysis, play a critical role in regulating the amplitude, duration and compartmentalization of cyclic nucleotide signalling. Abnormal alterations of PDEs and subsequent changes in cyclic nucleotide homoeostasis have been implicated in a number of different diseases. In the study published in the latest issue of Clinical Science , Bautista Niño and colleagues have shown that, in cultured senescent human VSMCs, PDE1A and PDE1C mRNA levels are significantly up-regulated and inhibition of PDE1 activity with vinpocetine reduced cellular senescent makers in senescent VSMCs. Moreover, in the premature aging mice with genomic instability ( Ercc1 d/− ), impaired aortic ring relaxation in response to SNP (sodium nitroprusside), an NO (nitric oxide) donor, was also largely improved by vinpocetine. More interestingly, using data from human GWAS (genome-wide association studies), it has been found that PDE1A single nucleotide polymorphisms is significantly associated with diastolic blood pressure and carotid intima–media thickening, two hallmarks of human vascular dysfunction in aging. These findings establish a strong relationship between PDE1 expression regulation and vascular abnormalities in aging.

AngII (angiotensin II) is a potent neurohormone responsible for cardiac hypertrophy, in which TGF (transforming growth factor)-β serves as a principal downstream mediator. We recently found that ablation of fibulin-2 in mice attenuated TGF-β signalling, protected mice against progressive ventricular dysfunction, and significantly reduced the mortality after experimental MI (myocardial infarction). In the present study, we investigated the role of fibulin-2 in AngII-induced TGF-β signalling and subsequent cardiac hypertrophy. We performed chronic subcutaneous infusion of AngII in fibulin-2 null (Fbln2 −/− ), heterozygous (Fbln2 +/− ) and WT (wild-type) mice by a mini-osmotic pump. After 4 weeks of subpressor dosage of AngII infusion (0.2 μg/kg of body weight per min), WT mice developed significant hypertrophy, whereas the Fbln2 −/− showed no response. In WT, AngII treatment significantly up-regulated mRNAs for fibulin-2, ANP (atrial natriuretic peptide), TGF-β1, Col I (collagen type I), Col III (collagen type III), MMP (matrix metalloproteinase)-2 and MMP-9, and increased the phosphorylation of TGF-β-downstream signalling markers, Smad2, TAK1 (TGF-β-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase), which were all unchanged in AngII-treated Fbln2 −/− mice. The Fbln2 +/− mice consistently displayed AngII-induced effects intermediate between WT and Fbln2 −/− . Pressor dosage of AngII (2 mg/kg of body weight per min) induced significant fibrosis in WT but not in Fbln2 −/− mice with comparable hypertension and hypertrophy in both groups. Isolated CFs (cardiac fibroblasts) were treated with AngII, in which direct AngII effects and TGF-β-mediated autocrine effects was observed in WT. The latter effects were totally abolished in Fbln2 −/− cells, suggesting that fibulin-2 is essential for AngII-induced TGF-β activation. In conclusion our data indicate that fibulin-2 is essential for AngII-induced TGF-β-mediated cardiac hypertrophy via enhanced TGF-β activation and suggest that fibulin-2 is a potential therapeutic target to inhibit AngII-induced cardiac remodelling.

FXIII (Factor XIII) is a Ca 2+ -dependent enzyme which forms covalent ϵ-(γ-glutamyl)lysine cross-links between the γ-carboxy-amine group of a glutamine residue and the ϵ-amino group of a lysine residue. FXIII was originally identified as a protein involved in fibrin clot stabilization; however, additional extracellular and intracellular roles for FXIII have been identified which influence thrombus resolution and tissue repair. The present review discusses the substrates of FXIIIa (activated FXIII) involved in thrombosis and wound healing with a particular focus on: (i) the influence of plasma FXIIIa on the formation of stable fibrin clots able to withstand mechanical and enzymatic breakdown through fibrin–fibrin cross-linking and cross-linking of fibrinolysis inhibitors, in particular α 2 -antiplasmin; (ii) the role of intracellular FXIIIa in clot retraction through cross-linking of platelet cytoskeleton proteins, including actin, myosin, filamin and vinculin; (iii) the role of intracellular FXIIIa in cross-linking the cytoplasmic tails of monocyte AT 1 Rs (angiotensin type 1 receptors) and potential effects on the development of atherosclerosis; and (iv) the role of FXIIIa on matrix deposition and tissue repair, including cross-linking of extracellular matrix proteins, such as fibronectin, collagen and von Willebrand factor, and the effects on matrix deposition and cell–matrix interactions. The review highlights the central role of FXIIIa in the regulation of thrombus stability, thrombus regulation, cell–matrix interactions and wound healing, which is supported by observations in FXIII-deficient humans and animals.

Varicose vein disease is a frequently occurring pathology with multifactorial causes and a genetic component. An intense remodelling of the varicose vein wall has been described and could be at the origin of its weakness and altered elasticity. We have described previously a dysregulation of collagen synthesis in cultured smooth muscle cells from saphenous veins and in dermal fibroblasts from the skin of patients with varicose veins, suggesting a systemic defect in their connective tissue. The present study describes comparative morphological and immunohistochemical data in both the skin and saphenous veins of eight control subjects (undergoing coronary bypass surgery) and eight patients with varicose veins. Histological staining of glycoproteins, the elastic fibre network and collagen bundles showed that the remodelling and fragmentation of elastic fibres observed in varicose veins were also present in the skin of the patients. When compared with control subjects, we observed in both the veins and skin of patients with varicose veins (i) an increase in the elastic network, as quantified by image analysis; (ii) an accumulation of collagen type I, fibrillin-1 and laminin; and (iii) an overproduction of MMP (matrix metalloproteinase)-1, MMP-2 and MMP-3, analysed by immunohistochemistry, but normal levels of other MMPs (MMP-7 and MMP-9) and their inhibitors (TIMP-1, TIMP-2 and TIMP-3). An imbalance of extracellular matrix production/degradation was thus observed in veins as well as in the skin of the patients with varicose veins and, taken together, these findings show that remodelling is present in different organs, confirming systemic alterations of connective tissues.

Abdominal aortic aneurysm (AAA) is characterized by chronic aortic wall inflammation and loss of matrix components. Proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) are thought to be involved in this inflammatory process and, therefore, to play an important role in the pathogenesis of human AAA. TNF-α-converting enzyme (TACE) has recently been purified and cloned as a disintegrin and metalloproteinase that converts TNF-α precursor into its mature form. The aim of the present study was to determine whether TNF-α and TACE were expressed and localized in aortic tissues in human AAA. Infrarenal aortic tissues were obtained from AAA patients ( n =19) undergoing elective aneurysm reconstruction and from autopsy cases without cardiovascular disorders as normal controls ( n =5). Internal thoracic artery samples were also obtained from patients with coronary artery disease undergoing coronary artery bypass grafting to represent biopsied conduit vessels ( n =5). The AAA specimens were taken from the mid-portion of the aneurysm and from the longitudinal transition zone between the non-dilated aorta and the proximal aspect of the aneurysm. TNF-α and TACE mRNA levels were determined by real-time quantitative reverse transcriptase–PCR. Expression levels of both TNF-α mRNA and TACE mRNA were significantly greater in the transition zone than in the mid-portion (both P <0.05). Expression levels of both forms of mRNA were significantly higher in AAA samples than in control aortas or atherosclerotic arteries. There was a significant correlation between the expression of TNF-α mRNA with that of TACE mRNA in AAA ( r =0.54, P <0.005). Immunostaining was positive for both TNF-α and TACE in CD68-positive macrophages in the media and adventitia obtained from the transition zone in AAA, whereas neither TNF-α nor TACE was expressed in control vessels. In conclusion, the concomitant activation and localization of TNF-α and TACE in the media and adventitia of the transition zone in human AAA underlines the importance of this system in the pathogenesis of this disorder.

1. The efficient repair of gastrointestinal mucosal injuries is essential in the preservation of the epithelial barrier to luminal antigens. Accumulated evidence suggests that epithelial migration plays a major part in this repair by rapidly resealing defects induced by both physiological and pathological insults, a process termed restitution. 2. This migration has been modelled in various ways, most commonly in mechanically wounded monolayers of cell lines or cells in primary culture, and in wounded human or animal tissue. Evidence from these models indicates that migration is a highly complex process, which is likely to involve the tightly controlled spatial and temporal interaction of multiple factors: (i) extracellular molecules such as soluble factors (e.g. growth factors, trefoil peptides, cytokines) and matrix components (e.g. collagen, laminin, fibronectin); (ii) signalling molecules activated by the interaction of these factors with cell surface receptors (e.g. protein kinases, phospholipases, low-molecular-weight GTPases); (iii) factors which regulate adhesion to other cells (e.g. E-cadherin) and to matrix components (e.g. integrins, hyaluronic acid receptors); (iv) factors which regulate detachment from the extracellular matrix (e.g. urokinase-type plasminogen activator, matrix metalloproteinases); and (iv) molecules which regulate cytoskeletal function (e.g. Rac), which allows the formation of specialized cellular processes termed lamellipodia. 3. The identification of physiologically relevant factors that stimulate epithelial cell migration, and a better understanding of their mechanism of action, may be beneficial in the development of novel therapeutic approaches in diseases such as inflammatory bowel disease through the pharmacological or dietary enhancement of this migration.

1. Multiple sclerosis is characterized by areas of demyelination spread throughout the central nervous system, in which the myelin sheaths surrounding axons are destroyed. While therapies aimed at suppressing the autoimmune response, such as β-interferon, may prevent further damage, they cannot repair or replace the lost myelin. To this end, an additional therapy has been proposed, which involves transplanting cells of the oligodendrocyte lineage into the central nervous system. 2. The cell of interest for transplantation is the oligodendrocyte precursor because, unlike the differentiated cell, it is an intrinsically migratory and proliferative cell. In order to optimize the transplant strategy we have investigated the molecular mechanisms that control migration in vitro , so that these mechanisms might be upregulated to maximize cell migration in vivo. We have focused on the integrin family of cell adhesion molecules, known to play a fundamental role in the regulation of migration in other cell types. 3. These studies show that oligodendrocytes express a limited repertoire of integrins consisting of α 6 β 1 and three different α v integrins. α 6 β 1 is expressed throughout development but α v integrins show developmental regulation; differentiation is accompanied by loss of α v β 1 and upregulation of α v β 5 . 4. Function-blocking studies show that oligodendrocyte precursor migration in vitro is mediated primarily by the developmentally regulated α v β 1 integrin, but not α 6 β 1 or α v β 3 . Taken together with previous evidence that cell migration can be regulated by altering integrin expression, this work suggests that modifying expression levels of α v β 1 on oligodendrocyte precursors may increase the migratory capacity of these cells. If so, this would support a future therapeutic strategy aimed at transplanting genetically modified oligodendrocyte precursors to repair widespread demyelinated lesions.

1. The present study was carried out to determine how levels of the mRNA of metalloproteinases (metalloproteinase-1, 72 kDa type IV collagenase, metalloproteinase-3 and 92 kDa type IV collagenase) and tissue inhibitor of metalloproteinases are regulated in the renal tissues of New Zealand Black/White F 1 mice. 2. mRNA levels for metalloproteinase-1, 72 kDa type IV collagenase, metalloproteinase-3 and tissue inhibitor of metalloproteinases increased significantly with the progression of nephritis in New Zealand Black/White F 1 mice. 3. At 48 weeks of age, the levels of mRNA for metalloproteinase-1, 72 kDa type IV collagenase, metalloproteinase-3 and tissue inhibitor of metalloproteinases increased by 8-, 4-, 8- and 15-fold, respectively, in the renal tissues of New Zealand Black/White F 1 mice compared with New Zealand White mice. 4. In the kidneys of New Zealand White mice, however, the mRNA levels for these proteins changed little throughout the experimental period. 5. We could not detect expression of mRNA for 9 2 kDa type IV collagenase in the renal tissue of New Zealand Black/White F 1 mice at 8 weeks of age or in New Zealand White mice at 8, 24 or 48 weeks of age, whereas we could detect expression of mRNA for this protein in New Zealand Black/White F 1 mice at 24 and 48 weeks of age when mononuclear cells had infiltrated the interstitium and surrounding blood vessels. 6. At 24 weeks of age, New Zealand Black/White F 1 mice were divided into two groups and received either methylprednisolone or saline injection for 24 weeks. 7. The development of histopathological lesions and increases in mRNA for metalloproteinases and tissue inhibitor of metalloproteinases were suppressed by treatment with methylprednisolone. 8. These data suggest that abnormal regulation of the genes for metalloproteinases and tissue inhibitor of metalloproteinases contribute to the accumulation of extracellular matrix components in lupus nephritis and that the beneficial effect of methylprednisolone is associated with its ability to suppress the expression of mRNA for metalloproteinases and tissue inhibitor of metalloproteinases.