FXR agonists INT-787 and OCA increase RECK and inhibit liver steatosis and inflammation in diet-induced ob/ob mouse model of NASH.

BACKGROUND AND AIMS: We have previously shown in a model of hepatic ischaemia/reperfusion injury that the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) restores reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), an inverse modulator of metalloproteases (MMPs) and inhibitor of the sheddases ADAM10 and ADAM17 involved in inflammation and fibrogenesis. Here, the effects of FXR agonists OCA and INT-787 on hepatic levels of RECK, MMPs, ADAM10 and ADAM17 were compared in a diet-induced ob/ob mouse model of non-alcoholic steatohepatitis (NASH). METHODS: Lep ob/ob NASH mice fed a high-fat diet (HFD) or control diet (CD) for 9 weeks (wks) were treated with OCA or INT-787 0.05% dosed via HFD admixture (30 mg/kg/day) or HFD for further 12 wks. Serum alanine transaminase (ALT) and inflammatory cytokines, liver RECK, MMP-2 and MMP-9 activity as well as ADAM10, ADAM17, collagen deposition (Sirius red), hepatic stellate cell activation (α-SMA) and pCK+ reactive biliary cells were quantified. RESULTS: Only INT-787 significantly reduced serum ALT, IL-1ß and TGF-ß. A downregulation of RECK expression and protein levels observed in HFD groups (at 9 and 21 wks) was counteracted by both OCA and INT-787. HFD induced a significant increase in liver MMP-2 and MMP-9; OCA administration reduced both MMP-2 and MMP-9 while INT-787 markedly reduced MMP-2 expression. OCA and INT-787 reduced both ADAM10 and ADAM17 expression and number of pCK+ cells. INT-787 was superior to OCA in decreasing collagen deposition and α-SMA levels. CONCLUSION: INT-787 is superior to OCA in controlling specific cell types and clinically relevant anti-inflammatory and antifibrotic molecular mechanisms in NASH.

Recent Updates on the Therapeutic Prospects of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs (RECK) in Liver Injuries.

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored glycoprotein, negatively regulates various membrane proteins involved in the tissue governing extracellular matrix (ECM) remodeling such as metalloproteases (MMPs) and the sheddases ADAM10 and ADAM17. The significance of the present review is to summarize the current understanding of the pathophysiological role of RECK, a newly discovered signaling pathway associated with different liver injuries. Specifically, this review analyzes published data on the downregulation of RECK expression in hepatic ischemia/reperfusion (I/R) injury, liver-related cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), as well as in the progression of nonalcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). In addition, this review discusses the regulation of RECK by inducers, such as FXR agonists. The RECK protein has also been suggested as a potential diagnostic and prognostic marker for liver injury or as a biomarker with predictive value for drug treatment efficacy.

Fatty Acids and Bilirubin as Intrinsic Autofluorescence Serum Biomarkers of Drug Action in a Rat Model of Liver Ischemia and Reperfusion.

The autofluorescence of specific fatty acids, retinoids, and bilirubin in crude serum can reflect changes in liver functional engagement in maintaining systemic metabolic homeostasis. The role of these fluorophores as intrinsic biomarkers of pharmacological actions has been investigated here in rats administered with obeticholic acid (OCA), a Farnesoid-X Receptor (FXR) agonist, proven to counteract the increase of serum bilirubin in hepatic ischemia/reperfusion (I/R) injury. Fluorescence spectroscopy has been applied to an assay serum collected from rats submitted to liver I/R (60/60 min ± OCA administration). The I/R group showed changes in the amplitude and profiles of emission spectra excited at 310 or 366 nm, indicating remarkable alterations in the retinoid and fluorescing fatty acid balance, with a particular increase in arachidonic acid. The I/R group also showed an increase in bilirubin AF, detected in the excitation spectra recorded at 570 nm. OCA greatly reversed the effects observed in the I/R group, confirmed by the biochemical analysis of bilirubin and fatty acids. These results are consistent with a relationship between OCA anti-inflammatory effects and the acknowledged roles of fatty acids as precursors of signaling agents mediating damaging responses to harmful stimuli, supporting serum autofluorescence analysis as a possible direct, real-time, cost-effective tool for pharmacological investigations.

Autofluorescence Label-Free Imaging of the Liver Reticular Structure.

Autofluorescence rising from biological substrates under proper excitation light depends on the presence of specific endogenous fluorophores and can provide information on the morpho-functional properties in which they are strictly involved. Besides the numerous endogenous fluorophores involved in metabolic functions, fibrous proteins may act as direct, label-free biomarkers of the tissue structural organization. The optical properties of collagen, in particular, are currently applied as an alternative to established histochemical procedures to investigate the connective tissue as well as its changes in diseased conditions. This is particularly true in hepatology where the histochemical procedures to label the reticular structure are not routinely applied, as they are complex and time-consuming. The morphology of the liver reticular structure and its changes are up to now poorly considered despite the increasing awareness of the regulatory role played by the remodeling of the reticular structure in pathological conditions. In this context, the autofluorescence label-free imaging has proven to be a suitable approach.

MCD Diet Rat Model Induces Alterations in Zinc and Iron during NAFLD Progression from Steatosis to Steatohepatitis.

We evaluate the effects of the methionine-choline-deficient (MCD) diet on serum and hepatic zinc (Zn) and iron (Fe) and their relationships with matrix metalloproteinases (MMPs) and their modulators (TIMPs and RECK) as well as hepatic fatty acids using male Wistar rats fed 2-, 4- and 8-week MCD diets. Serum and hepatic Zn decrease after an 8-week MCD diet. Serum Fe increases after an 8-week MCD diet and the same occurs for hepatic Fe. An increase in hepatic MMP activity, associated with a decrease in RECK and TIMPs, is found in the MCD 8-week group. Liver Fe shows a positive correlation versus MMPs and RECK, and an inverse correlation versus TIMPs. A positive correlation is found comparing liver Zn with stearic, vaccenic and arachidonic acids, and an inverse correlation is found with linolenic and docosatetraenoic acids. An opposite trend is found between liver Fe versus these fatty acids. During NAFLD progression from steatosis to steatohepatitis, MCD rats exhibit an increase in Zn and a decrease in Fe levels both in serum and tissue associated with alterations in hepatic MMPs and their inhibitors, and fatty acids. The correlations detected between Zn and Fe versus extracellular matrix modulators and fatty acids support their potential role as therapeutic targets.

Obeticholic Acid Reduces Kidney Matrix Metalloproteinase Activation Following Partial Hepatic Ischemia/Reperfusion Injury in Rats.

We have previously demonstrated that the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) protects the liver via downregulation of hepatic matrix metalloproteinases (MMPs) after ischemia/reperfusion (I/R), which can lead to multiorgan dysfunction. The present study investigated the capacity of OCA to modulate MMPs in distant organs such as the kidney. Male Wistar rats were dosed orally with 10 mg/kg/day of OCA (5 days) and were subjected to 60-min partial hepatic ischemia. After 120-min reperfusion, kidney biopsies (cortex and medulla) and blood samples were collected. Serum creatinine, kidney MMP-2, and MMP-9-dimer, tissue inhibitors of MMPs (TIMP-1, TIMP-2), RECK, TNF-alpha, and IL-6 were monitored. MMP-9-dimer activity in the kidney cortex and medulla increased after hepatic I/R and a reduction was detected in OCA-treated I/R rats. Although not significantly, MMP-2 activity decreased in the cortex of OCA-treated I/R rats. TIMPs and RECK levels showed no significant differences among all groups considered. Serum creatinine increased after I/R and a reduction was detected in OCA-treated I/R rats. The same trend occurred for tissue TNF-alpha and IL-6. Although the underlying mechanisms need further investigation, this is the first study showing, in the kidney, beneficial effects of OCA by reducing TNF-alpha-mediated expression of MMPs after liver I/R.

Associations between serum trace elements and inflammation in two animal models of nonalcoholic fatty liver disease.

BACKGROUND: The comparison of hepatic steatosis animal models has allowed the understanding of mechanisms involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and the progression to nonalcoholic steatohepatitis (NASH). We investigated the changes in serum levels of trace elements and inflammation markers in fatty livers using two rat models of NAFLD, the methionine and choline deficient (MCD) diet model and Obese-Zucker rats. MATERIAL AND METHODS: NAFLD was induced in male Wistar rats by 3-week MCD diet administration, after which, blood samples were collected. 12-week old Obese (fa/fa) and Lean (fa/-) male Zucker rats were also used. Serum levels of hepatic enzymes, Urea, Uric acid, Ca2+, Cl, Fe, K, Na, Mg and Zn were quantified, as well as the inflammation markers TNF-alpha, IL-1beta and IL-6. RESULTS: In MCD rats, a serum increase in Cl, Mg and Na and a decrease in Ca2+, Zn were detected in comparison with control rats. An increase in only serum Ca2+ was found in Obese-Zucker rats. In MCD rat serum, Zn was inversely correlated with IL-1beta, IL-6, TNF-alpha, Urea and Uric Acid; Ca2+ was inversely correlated with IL-1beta, IL-6 and Urea; Cl and Mg were directly correlated with Uric Acid and Urea, respectively. In Obese-Zucker rats, Cl and IL-1beta were inversely correlated, whereas Ca2+ and Urea where directly correlated, as well Fe and TNF-alpha. CONCLUSIONS: The serum concentrations of trace elements change significantly only in MCD rats, which spontaneously progress to NASH. The causes of these changes may be a result of defense strategies of the organism, which is regulated by immunoregulatory cytokines. These results might suggest that the impairment of trace element status should be taken into account when the effectiveness of a pharmacological treatment is under evaluation.

Obeticholic acid reduces biliary and hepatic matrix metalloproteinases activity in rat hepatic ischemia/reperfusion injury.

BACKGROUND: We have previously shown that obeticholic acid (OCA) upregulates the biliary excretion of asymmetric dimethylarginine (ADMA), an inhibitor of iNOS regulating the activity of matrix metalloproteinases (MMPs). Here, the effects of OCA on MMP-2 and MMP-9 activity in liver, bile and serum were evaluated after hepatic ischemia/reperfusion (I/R) injury. MATERIAL AND METHODS: Male Wistar rats (n = 20) were orally administered 10 mg/kg/day of OCA (5 days) and subjected to a 60-min ischemia and 60-min reperfusion. Bile, serum and tissue were collected for MMP-2 and MMP-9 activity quantification. The MMP regulator tissue reversion-inducing cysteine rich protein with Kazal motifs (RECK), tissue inhibitor of metalloproteinases (TIMPs), iNOS and biliary levels of LDH, γGT, glucose and ADMA were quantified. RESULTS: In the I/R group, OCA administration reduced MMP-2 and MMP-9 in liver, bile and serum. A downregulation of tissue RECK and TIMPs, observed under I/R, were recovered by OCA. Immunohistochemical staining of hepatic tissue demonstrated that RECK expression is mainly localized in both cholangiocytes and hepatocytes. Hepatic iNOS positively correlated with tissue MMP-2 and MMP-9 activity. Biliary levels of LDH, γGT and glucose were lower in I/R rats treated with OCA; in bile, MMP levels positively correlated with LDH and γGT. CONCLUSION: Thus, OCA administration confers protection to cholangiocytes via downregulation of biliary MMPs in livers submitted to I/R. This event is associated with hepatic RECK- and TIMP-mediated MMP decrease.

Transient Expression of Reck Under Hepatic Ischemia/Reperfusion Conditions Is Associated with Mapk Signaling Pathways.

In this study, we demonstrated the involvement of matrix metalloproteinases (MMPs) in hepatic ischemia/reperfusion (I/R) injury. Our aim is to evaluate the impact of reperfusion on I/R-related changes in RECK, an MMP modulator, and mitogen-activated protein kinase (MAPKs) pathways (ERK, p38, and JNK). Male Wistar rats were either subjected to 60 min partial-hepatic ischemia or sham-operated. After a 60 min or 120 min reperfusion, liver samples were collected for analysis of MMP-2 and MMP-9 by zymography and RECK, TIMP-1, and TIMP-2 content, MAPKs activation (ERK1/2, JNK1/2, and p38), as well as iNOS and eNOS by Western blot. Serum enzymes AST, ALT, and alkaline-phosphatase were quantified. A transitory decrease in hepatic RECK and TIMPs was associated with a transitory increase in both MMP-2 and MMP-9 activity and a robust activation of ERK1/2, JNK1/2, and p38 were detected at 60 min reperfusion. Hepatic expression of iNOS was maximally upregulated at 120 min reperfusion. An increase in eNOS was detected at 120 min reperfusion. I/R evoked significant hepatic injury in a time-dependent manner. These findings provide new insights into the underlying molecular mechanisms of reperfusion in inducing hepatic injury: a transitory decrease in RECK and TIMPs and increases in both MAPK and MMP activity suggest their role as triggering factors of the organ dysfunction.

Fatty Acid Desaturase Involvement in Non-Alcoholic Fatty Liver Disease Rat Models: Oxidative Stress Versus Metalloproteinases.

We investigated changes in fatty acid desaturases, D5D, D6D, D9-16D and D9-18D, and their relationship with oxidative stress, matrix metalloproteinases (MMPs) and serum TNF-alpha in two rat models of non-alcoholic fatty liver disease NAFLD. Eight-week-old male Wistar rats fed for 3 weeks with methionine-choline⁻deficient (MCD) diet and eleven-week-old Obese male Zucker rats were used. Serum levels of hepatic enzymes and TNF-alpha were quantified. Hepatic oxidative stress (ROS, TBARS and GSH content) and MMP-2 and MMP-9 (protein expression and activity) were evaluated. Liver fatty acid profiling, performed by GC-MS, was used for the quantification of desaturase activities. Higher D5D and D9-16D were found in Obese Zucker rats as well as an increase in D9-18D in MCD rats. D6D was found only in MCD rats. A negative correlation between D5D and D9-16D versus TBARS, ROS and TNF-alpha and a positive correlation with GSH were shown in fatty livers besides a positive correlation between D9-18D versus TBARS, ROS and TNF-alpha and a negative correlation with GSH. A positive correlation between D5D or D9-16D or D9-18D versus protein expression and the activity of MMP-2 were found. NAFLD animal models showed comparable serum enzymes. These results reinforce and extend findings on the identification of therapeutic targets able to counteract NAFLD disorder.

Animal Models of Steatosis (NAFLD) and Steatohepatitis (NASH) Exhibit Hepatic Lobe-Specific Gelatinases Activity and Oxidative Stress.

Animal models of obstructive cholestasis and ischemia/reperfusion damage have revealed the functional heterogeneity of liver lobes. This study evaluates this heterogeneity in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) rat models. Twelve-week-old Obese and Lean male Zucker rats were used for NAFLD. Eight-week-old male Wistar rats fed with 8-week methionine-choline-deficient (MCD) diet and relative control diet were used for NASH. Gelatinase (MMP-2; MMP-9) activity and protein levels, tissue inhibitors of metalloproteinase (TIMPs), reactive oxygen species (ROS), and thiobarbituric acid-reactive substances (TBARS) were evaluated in the left (LL), median (ML), and right liver (RL) lobes. Serum hepatic enzymes and TNF-alpha were assessed. An increase in gelatinase activity in the NASH model occurred in RL compared with ML. TIMP-1 and TIMP-2 displayed the same trend in RL as ML and LL. Control diet RL showed higher MMP-9 activity compared with ML and LL. No significant lobar differences in MMP-2 activity were detected in the NAFLD model. MMP-9 activity was not detectable in Zucker rats. TIMP-1 was lower in LL when compared with ML while no lobar differences were detectable for TIMP-2 in either Obese or Lean Zucker rats. Control diet rats exhibited higher ROS formation in LL versus RL. Significant increases in TBARS levels were observed in LL versus ML and RL in control and MCD rats. The same trend for ROS and TBARS was found in Obese and Lean Zucker rats. An increased serum TNF-alpha occurred in MCD rats. A lobar difference was detected for MMPs, TIMPs, ROS, and TBARS in both MCD and Zucker rats. Higher MMP activation in RL and higher oxidative stress in the LL, compared with the other lobes studied, supports growing evidence for functional heterogeneity among the liver lobes occurring certainly in both NAFLD and NASH rats.

Proteotoxicity in cardiac amyloidosis: amyloidogenic light chains affect the levels of intracellular proteins in human heart cells.

AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. Besides the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at cellular level are complex and largely undefined. We have characterized the molecular changes in primary human cardiac fibroblasts (hCFs) exposed in vitro to soluble amyloidogenic cardiotoxic LCs from AL cardiomyopathy patients. To evaluate proteome alterations caused by a representative cardiotropic LC, we combined gel-based with label-free shotgun analysis and performed bioinformatics and data validation studies. To assess the generalizability of our results we explored the effects of multiple LCs on hCF viability and on levels of a subset of cellular proteins. Our results indicate that exposure of hCFs to cardiotropic LCs translates into proteome remodeling, associated with apoptosis activation and oxidative stress. The proteome alterations affect proteins involved in cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. These results support and expand the concept that soluble amyloidogenic cardiotropic LCs exert toxic effects on cardiac cells.

Effects of a Bioavailable Arabinoxylan-enriched White Bread Flour on Postprandial Glucose Response in Normoglycemic Subjects.

The beneficial effects of soluble fibers on carbohydrate metabolism are well documented. In this regard, we tested an arabinoxylan-enriched white bread flour, obtained by a patented process by which the bran extracted from the milling process is enzymatically hydrolyzed in order to separate the soluble fraction fiber from the insoluble fiber. We recruited 24 healthy normoglycemic volunteers [Age 34-61 ± 12.5 y; Body Mass Index (BMI) 22.1 ± 2.5 kg/m(2); Waist circumference (WC) 84.43 ± 8.0 cm; Fat Mass (FM) 22.7 ± 8.0%] attending the Dietetics Outpatient Clinic of the Internal Medicine Department at IRCCS Policlinico S. Matteo Foundation, University of Pavia, Pavia, Italy. Subjects acutely consumed arabinoxylan-enriched white bread (weight: 100 g) or isoenergetic control breads, in a double-blind crossover study design. Plasma glucose levels were measured just before bread administration and 30 minutes afterwards. The 30-minute peak postprandial glucose concentrations after arabinoxylan-enriched meals were significantly lower than after the control meal (107±4.6 mg/dL vs. 121 ± 5.2 mg/dL; p < 0.05). The here-reported results show how postprandial glucose responses were improved by ingestion of the arabinoxylan-enriched meal. Further studies are needed to clarify whether daily consumption of arabinoxylan-enriched bread will benefit patients with type 2 diabetes mellitus.

Role of matrix metalloproteinases in cholestasis and hepatic ischemia/reperfusion injury: A review.

Matrix metalloproteinases (MMPs) are a family of proteases using zinc-dependent catalysis to break down extracellular matrix (ECM) components, allowing cell movement and tissue reorganization. Like many other proteases, MMPs are produced as zymogens, an inactive form, which are activated after their release from cells. Hepatic ischemia/reperfusion (I/R) is associated with MMP activation and release, with profound effects on tissue integrity: their inappropriate, prolonged or excessive expression has harmful consequences for the liver. Kupffer cells and hepatic stellate cells can secrete MMPs though sinusoidal endothelial cells are a further source of MMPs. After liver transplantation, biliary complications are mainly attributable to cholangiocytes, which, compared with hepatocytes, are particularly susceptible to injury and ultimately a major cause of increased graft dysfunction and patient morbidity. This paper focuses on liver I/R injury and cholestasis and reviews factors and mechanisms involved in MMP activation together with synthetic compounds used in their regulation. In this respect, recent data have demonstrated that the role of MMPs during I/R may go beyond the mere destruction of the ECM and may be much more complex than previously thought. We thus discuss the role of MMPs as an important factor in cholestasis associated with I/R injury.

Novel mitochondrial protein interactors of immunoglobulin light chains causing heart amyloidosis.

In immunoglobulin (Ig) light-chain (LC) (AL) amyloidosis, AL deposition translates into life-threatening cardiomyopathy. Clinical and experimental evidence indicates that soluble cardiotoxic LCs are themselves harmful for cells, by which they are internalized. Hypothesizing that interaction of soluble cardiotoxic LCs with cellular proteins contributes to damage, we characterized their interactome in cardiac cells. LCs were purified from patients with AL amyloidosis cardiomyopathy or multiple myeloma without amyloidosis (the nonamyloidogenic/noncardiotoxic LCs served as controls) and employed at concentrations in the range observed in AL patients' sera. A functional proteomic approach, based on direct and inverse coimmunoprecipitation and mass spectrometry, allowed identifying LC-protein complexes. Findings were validated by colocalization, fluorescence lifetime imaging microscopy (FLIM)-fluorescence resonance energy transfer (FRET), and ultrastructural studies, using human primary cardiac fibroblasts (hCFs) and stem cell-derived cardiomyocytes. Amyloidogenic cardiotoxic LCs interact in vitro with specific intracellular proteins involved in viability and metabolism. Imaging confirmed that, especially in hCFs, cardiotoxic LCs (not controls) colocalize with mitochondria and spatially associate with selected interactors: mitochondrial optic atrophy 1-like protein and peroxisomal acyl-coenzyme A oxidase 1 (FLIM-FRET efficiencies 11 and 6%, respectively). Cardiotoxic LC-treated hCFs display mitochondrial ultrastructural changes, supporting mitochondrial involvement. We show that cardiotoxic LCs establish nonphysiologic protein-protein contacts in human cardiac cells, offering new clues on the pathogenesis of AL cardiomyopathy.

Lobe-specific heterogeneity in asymmetric dimethylarginine and matrix metalloproteinase levels in a rat model of obstructive cholestasis.

We investigated the effects of obstructive cholestasis in different hepatic lobes by evaluating asymmetric dimethylarginine (ADMA) (a nitric oxide synthase inhibitor), protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (enzymes involved, resp., in its synthesis and degradation), the cationic transporter (CAT), and metalloproteinase (MMP) activity. Sixteen male Wistar rats underwent a 3-day cholestasis by common bile duct ligation (BDL) or sham operation. Blood samples and hepatic biopsies from left lobe (LL), median lobe (ML), and right lobe (RL) were collected. Serum hepatic enzymes, tissue ADMA, DDAH activity, CAT-2 protein, mRNA expression of DDAH and PRMT, and MMP-2 and MMP-9 activity were monitored. Cholestasis was confirmed by altered serum hepatic enzymes. Higher levels of tissue ADMA were detected in RL and ML as compared with LL. PRMT mRNA expression and DDAH activity did not differ among the lobes after BDL. CAT-2 levels are higher in the RL and ML in the sham-operated group. Higher activity in MMP-2 and MMP-9 was found in RL. In conclusion, after cholestasis an increase in hepatic ADMA in RL and ML was detected as well as tissue MMP-2 and MMP-9 activation in RL, supporting the evidence of functional heterogeneity among the liver lobes also occurring in an obstructive cholestasis model.

Lung matrix metalloproteinase activation following partial hepatic ischemia/reperfusion injury in rats.

PURPOSE: Warm hepatic ischemia-reperfusion (I/R) injury can lead to multiorgan dysfunction. The aim of the present study was to investigate whether acute liver I/R does affect the function and/or structure of remote organs such as lung, kidney, and heart via modulation of extracellular matrix remodelling. METHODS: Male Sprague-Dawley rats were subjected to 30 min partial hepatic ischemia by clamping the hepatic artery and the portal vein. After a 60 min reperfusion, liver, lung, kidney, and heart biopsies and blood samples were collected. Serum hepatic enzymes, creatinine, urea, Troponin I and TNF-alpha, and tissue matrix metalloproteinases (MMP-2, MMP-9), myeloperoxidase (MPO), malondialdehyde (MDA), and morphology were monitored. RESULTS: Serum levels of hepatic enzymes and TNF-alpha were concomitantly increased during hepatic I/R. An increase in hepatic MMP-2 and MMP-9 activities was substantiated by tissue morphology alterations. Notably, acute hepatic I/R affect the lung inasmuch as MMP-9 activity and MPO levels were increased. No difference in MMPs and MPO was observed in kidney and heart. CONCLUSIONS: Although the underlying mechanism needs further investigation, this is the first study in which the MMP activation in a distant organ is reported; this event is probably TNF-alpha-mediated and the lung appears as the first remote organ to be involved in hepatic I/R injury.

A multimarker study of degenerative aortic valve disease: stenoinsufficiency shows more indices of bad prognosis.

OBJECTIVES: It was the aim of this study to assess the pathophysiological, prognostic role of aortic regurgitation (AR) in the 'mixed pictures' of degenerative aortic valve stenoinsufficiency (ASI) by a multimarker clinical approach. METHODS: We enrolled 112 consecutive surgical PATIENTS: 19 with pure valve stenosis (PAS), 39 with mild regurgitation, 29 with severe regurgitation, and 25 controls with annulo-ectatic AR. All underwent complete echocardiography, carotid ultrasound and aortic/coronary multislice computed tomography calcium score evaluation. We determined tissue semiquantitative osteopontin, metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and circulating brain natriuretic peptide. We evaluated major adverse cardiac events and cardiovascular early, long-term mortality after bioprosthetic valve implantation. RESULTS: Tissue calcification, carotid and coronary atherosclerotic disease were prevalent in PAS versus ASI and AR patients. The multislice computed tomography calcium score (Agatston) was comparable between PAS and ASI (PAS 3,507.3 + 2,442.6; mild AR 4,270.7 + 2,213.5; severe AR 3,568.5 + 1,823.4), but much lower in AR (1,247.8 + 2,708.6). In ASI, a plasma/tissue 'profibrotic' MMP/TIMP balance prevailed, with circulating and echocardiographic indices of myocardial dysfunction. Percentages of major adverse cardiac events and early, long-term mortality were higher in ASI. CONCLUSIONS: In ASI, different, still unknown, genetic and dysplastic factors could work synergically with cardiovascular risk factors, determining a much more adverse myocardial and valve remodeling, resulting in worse clinical outcome.

Lobe-specific heterogeneity and matrix metalloproteinase activation after ischemia/reperfusion injury in rat livers.

Studies assessing the effects of partial-hepatic ischemia/reperfusion (I/R) injury focused on the damage to the ischemic-lobe, whereas few data are available on non-ischemic lobe. This study investigated whether acute liver I/R does affect non-ischemic lobe function via modulation of extracellular matrix remodeling. Male Sprague-Dawley rats underwent left lateral- and median-lobe ischemia for 30 min and reperfusion for 60 min or sham operation. After reperfusion, blood samples and hepatic biopsies from both the ischemic (left-lobe, LL) and the non-ischemic lobe (right-lobe, RL) were collected. Serum hepatic enzymes and TNF-alpha, tissue matrix metalloproteinases (MMP-2, MMP-9), liver morphology, malondialdehyde (MDA), and myeloperoxidase (MPO) were evaluated. Liver I/R injury was confirmed by altered increased hepatic enzymes and TNF-alpha. I/R induced an altered morphology and an increase in MMP-2 and MMP-9 activity not only in left-ischemic lobe (LL) but also in the right-non-ischemic (RL) lobe. A lobar difference was detected for MDA formation and MPO activity in both sham and I/R submitted rats, with higher levels in the left lobe for both groups. This study indicates that an increase in MMPs, which may be TNF-alpha-mediated, occurs in both the ischemic- and the non-ischemic lobes; the heterogeneous lobe concentrations of MDA and MPO suggest that the random sampling of liver tissue should be avoided.

Distinct mechanisms for diastolic dysfunction in diabetes mellitus and chronic pressure-overload.

Chronic pressure-overload and diabetes mellitus are two frequent disorders affecting the heart. We aimed to characterize myocardial structural and functional changes induced by both conditions. Pressure-overload was established in Wistar-han male rats by supra-renal aortic banding. Six-weeks later, diabetes was induced by streptozotocin (65 mg/kg,ip), resulting in four groups: SHAM, banding (BA), diabetic (DM) and diabetic-banding (DB). Six-weeks later, pressure-volume loops were obtained and left ventricular samples were collected to evaluate alterations in insulin signalling pathways, extracellular matrix as well as myofilament function and phosphorylation. Pressure-overload increased cardiomyocyte diameter (BA 22.0 ± 0.4 µm, SHAM 18.2 ± 0.3 µm) and myofilament maximal force (BA 25.7 ± 3.6 kN/m(2), SHAM 18.6 ± 1.4 kN/m(2)), Ca(2+) sensitivity (BA 5.56 ± 0.02, SHAM 5.50 ± 0.02) as well as MyBP-C, Akt and Erk phosphorylation, while decreasing rate of force redevelopment (K (tr); BA 14.9 ± 1.1 s(-1), SHAM 25.2 ± 1.5 s(-1)). At the extracellular matrix level, fibrosis (BA 10.8 ± 0.9%, SHAM 5.3 ± 0.6%), pro-MMP-2 and MMP-9 activities increased and, in vivo, relaxation was impaired (τ; BA 14.0 ± 0.9 ms, SHAM 12.9 ± 0.4 ms). Diabetes increased cardiomyocyte diameter, fibrosis (DM 21.4 ± 0.4 µm, 13.9 ± 1.8%, DB 20.6 ± 0.4 µm, 13.8 ± 0.8%, respectively), myofilament Ca(2+)sensitivity (DM 5.57 ± 0.02, DB 5.57 ± 0.01), advanced glycation end-product deposition (DM 4.9 ± 0.6 score/mm(2), DB 5.1 ± 0.4 score/mm(2), SHAM 2.1 ± 0.3 score/mm(2)), and apoptosis, while decreasing K (tr) (DM 13.5 ± 1.9 s(-1), DB 15.2 ± 1.4 s(-1)), Akt phosphorylation and MMP-9/TIMP-1 and MMP-1/TIMP-1 ratios. Diabetic hearts were stiffer (higher end-diastolic-pressure: DM 7.0 ± 1.2 mmHg, DB 6.7 ± 0.7 mmHg, SHAM 5.3 ± 0.4 mmHg, steeper end-diastolic-pressure-volume relation: DM 0.59 ± 0.18, DB 0.83 ± 0.17, SHAM 0.41 ± 0.10), and hypo-contractile (decreased end-systolic-pressure-volume-relation). DB animals presented further pulmonary congestion (Lungs/body-weight: DB 5.23 ± 0.21 g/kg, SHAM 3.80 ± 0.14 g/kg) as this group combined overload-induced relaxation abnormalities and diabetes-induced stiffness. Diabetes mellitus and pressure overload led to distinct diastolic dysfunction phenotypes: while diabetes promoted myocardial stiffening, pressure overload impaired relaxation. The association of these damages accelerates the progression of diastolic heart failure progression in diabetic-banded animals.