metallothionein and Fibrosis

Venous leg ulcers (VLU) represent a major clinical unmet need, impairing quality of life for millions worldwide. The bioengineered bilayered living cell construct (BLCC) is the only FDA-approved therapy demonstrating efficacy in healing chronic VLU, yet its in vivo mechanisms of action are not well understood. Previously, we reported a BLCC-mediated acute wounding response at the ulcer edge; in this study we elucidated the BLCC-specific effects on the epidermis-free ulcer bed. We conducted a randomized controlled clinical trial (ClinicalTrials.gov NCT01327937) enrolling 30 subjects with nonhealing VLUs, and performed genotyping, genomic profiling, and functional analysis on wound bed biopsies obtained at baseline and 1 week after treatment with BLCC plus compression or compression therapy (control). The VLU bed transcriptome featured processes of chronic inflammation and was strikingly enriched for fibrotic/fibrogenic pathways and gene networks. BLCC application decreased expression of profibrotic TGFß1 gene targets and increased levels of TGFß inhibitor decorin. Surprisingly, BLCC upregulated metallothioneins and fibroblast-derived MMP8 collagenase, and promoted endogenous release of MMP-activating zinc to stimulate antifibrotic remodeling, a novel mechanism of cutaneous wound healing. By activating a remodeling program in the quiescent VLU bed, BLCC application shifts nonhealing to healing phenotype. As VLU bed fibrosis correlates with poor clinical healing, findings from this study identify the chronic VLU as a fibrotic skin disease and are first to support the development and application of antifibrotic therapies as a successful treatment approach.

Topics: Adult; Aged; Aged, 80 and over; Collagen; Compression Bandages; Decorin; Female; Fibrosis; Gene Expression Profiling; Humans; Inflammation; Male; Matrix Metalloproteinase 8; Metallothionein; Middle Aged; Phenotype; Skin, Artificial; Transforming Growth Factor beta1; Treatment Outcome; Varicose Ulcer; Wound Healing; Zinc

Topics: Adipokines; Animals; Apoptosis; Cell Movement; Cell Proliferation; Disease Models, Animal; Fibrosis; Heart; Hydrogen Peroxide; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Metallothionein; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardium; Signal Transduction; Superoxide Dismutase; Tumor Necrosis Factors; Ventricular Function, Left

As a main clinical feature of obstructive sleep apnea (OSA), intermittent hypoxia (IH) induces oxidative stress, leading to damage to a variety of organs, including kidney. Metallothionein (MT) is a potent antioxidant that protects kidney against oxidative damage. Our previous studies demonstrated that MT prevented IH-induced cardiomyopathy in mice. However, the role of MT in protecting against IH-induced renal injury is unknown. Therefore, MT knockout (MT KO) mice and wild type (WT) control mice (129S) were culled for exposure to intermittent air as control or IH for a time course of 3 days, 1 week, 3 weeks and 8 weeks. MT KO mice developed higher urinary albumin to creatinine ratio (UACR) after exposure to IH for 8 weeks. Compared with either MT KO control or WT IH mice, MT deletion significantly aggravated IH-induced renal oxidative damage and inflammation at all four time points, along with significant acceleration of renal fibrosis after exposure to IH for 3 weeks and 8 weeks. Antioxidants including MT, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase 1 (HO1) and NAD (P) H dehydrogenase [quinone] 1 (NQO1) were increased in response to short-term IH (3 days, 1 week and 3 weeks) but decreased after long-term IH (8 weeks) in WT mice. Interestingly, Nrf2, HO1 and NQO1 were significantly attenuated under IH conditions in the absence of MT, which were in parallel with the inactivation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK). These findings demonstrated that MT played a key role in preventing IH-induced renal injury possibly via preserving Nrf2 signaling pathway.

Topics: Acute Kidney Injury; Albuminuria; Animals; Antioxidants; Fibrosis; Gene Deletion; Hypoxia; Kidney Function Tests; Lipid Peroxidation; Metallothionein; Mice; Mice, Knockout; Nephritis

The development of diabetic cardiomyopathy is attributed to diabetic oxidative stress, which may be related to the mitogen-activated protein kinase (MAPK) c-Jun NH2-terminal kinase (JNK) activation. The present study tested a hypothesis whether the curcumin analog C66 [(2E,6E)-2,6-bis(2-(trifluoromethyl)benzylidene) cyclohexanone] as a potent antioxidant can protect diabetes-induced cardiac functional and pathogenic changes via inhibition of JNK function. Diabetes was induced with a single intraperitoneal injection of streptozotocin in male C57BL/6 mice. Diabetic and age-matched control mice were randomly divided into three groups, each group treated with C66, JNK inhibitor (JNKi, SP600125), or vehicle (1% CMC-Na solution) by gavage at 5 mg/kg every other day for 3 mo. Neither C66 nor JNKi impacted diabetic hyperglycemia and inhibition of body-weight gain, but both significantly prevented diabetes-induced JNK phosphorylation in the heart. Compared with basal line, cardiac function was significantly decreased in diabetic mice at 3 mo of diabetes but not in C66- or JNKi-treated diabetic mice. Cardiac fibrosis, oxidative damage, endoplasmic reticulum stress, and cell apoptosis, examined by Sirius red staining, Western blot, and thiobarbituric acid assay, were also significantly increased in diabetic mice, all which were prevented by C66 or JNKi treatment under diabetic conditions. Cardiac metallothionein expression was significantly decreased in diabetic mice but was almost normal in C66- or JNKi-treated diabetic mice. These results suggest that, like JNKi, C66 is able to prevent diabetic upregulation of JNK function, resulting in a prevention of diabetes-induced cardiac fibrosis, oxidative stress, endoplasmic reticulum stress, and cell death, along with a preservation of cardiac metallothionein expression.

Topics: Animals; Apoptosis; Benzylidene Compounds; Blood Pressure; Blotting, Western; Coloring Agents; Curcumin; Cyclohexanones; Diabetes Mellitus, Experimental; Diabetic Cardiomyopathies; Echocardiography; Endoplasmic Reticulum Stress; Fibrosis; Heart; JNK Mitogen-Activated Protein Kinases; Lipid Peroxidation; Male; Metallothionein; Mice; Mice, Inbred C57BL; Myocardium; Oxidative Stress; Phosphorylation; Protein Kinase Inhibitors; Real-Time Polymerase Chain Reaction

Second hand cigarette smoke is an independent risk factor for cardiovascular disease. Although a tie between smoking and cardiovascular disease is well established, the underlying mechanisms still remains elusive due to the lack of adequate animal models. This study was designed to use a mouse model of exposure to cigarette smoke, a surrogate of environmental tobacco smoke, to evaluate the impact of cardiac overexpression of heavy metal scavenger metallothionein on myocardial geometry, contractile and intracellular Ca(2+) properties and apoptosis following side-stream smoke exposure.. Adult male wild-type FVB and metallothionein transgenic mice were placed in a chamber exposed to cigarette smoke for 1 hour daily for 40 days. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, fibrosis, apoptosis and mitochondrial damage were examined.. Our data revealed that smoke exposure enlarged ventricular end systolic and diastolic diameters, reduced myocardial and cardiomyocyte contractile function, disrupted intracellular Ca(2+) homeostasis, facilitated fibrosis, apoptosis and mitochondrial damage (cytochrome C release and aconitase activity), the effects of which were attenuated or mitigated by metallothionein. In addition, side-stream smoke expose enhanced phosphorylation of Akt and GSK3β without affecting pan protein expression in the heart, the effect of which was abolished or ameliorated by metallothionein. Cigarette smoke extract interrupted cardiomyocyte contractile function and intracellular Ca(2+) properties, the effect of which was mitigated by wortmannin and NAC.. These data suggest that side-stream smoke exposure led to myocardial dysfunction, intracellular Ca(2+) mishandling, apoptosis, fibrosis and mitochondrial damage, indicating the therapeutic potential of antioxidant against in second smoking-induced cardiac defects possibly via mitochondrial damage and apoptosis.

Topics: Aconitate Hydratase; Animals; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Calcium; Calcium Signaling; Calcium-Binding Proteins; Cardiovascular Diseases; Cells, Cultured; Cytochromes c; Fibrosis; Male; Metallothionein; Mice; Mice, Transgenic; Mitochondria, Heart; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Nitric Oxide Synthase Type III; Organ Specificity; Protein Processing, Post-Translational; Reactive Oxygen Species; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Smoking; Tobacco Smoke Pollution

Cold exposure is associated with an increased prevalence of cardiovascular disease although the mechanism is unknown. Metallothionein, a heavy-metal-scavenging antioxidant, protects against cardiac anomalies. This study was designed to examine the impact of metallothionein on cold exposure-induced myocardial dysfunction, intracellular Ca(2+) derangement, fibrosis, endoplasmic reticulum (ER) stress, and apoptosis. Echocardiography, cardiomyocyte function, and Masson trichrome staining were evaluated in Friend virus B (FVB) and cardiac-specific metallothionein transgenic mice after cold exposure (3 months, 4 °C). Cold exposure increased plasma levels of norepinephrine, endothelin-1, and TGF-β; reduced plasma NO levels and cardiac antioxidant capacity; enlarged ventricular end-systolic diameter; compromised fractional shortening; promoted reactive oxygen species (ROS) production and apoptosis; and suppressed the ER stress markers Bip, calregulin, and phospho-eIF2α, accompanied by cardiac fibrosis and elevated levels of matrix metalloproteinases and Smad-2/3 in FVB mice. Cold exposure-induced echocardiographic, histological, ER stress, ROS, apoptotic, and fibrotic signaling changes (but not plasma markers) were greatly improved by metallothionein. In vitro metallothionein induction by zinc chloride ablated H(2)O(2)- but not TGF-β-induced cell proliferation in fibroblasts. In summary, our data suggest that metallothionein protects against cold exposure-induced cardiac anomalies possibly through attenuation of myocardial fibrosis.

Topics: Animals; Apoptosis; Calcium; Cold Temperature; Endoplasmic Reticulum Stress; Endothelin-1; Fibroblasts; Fibrosis; Friend murine leukemia virus; Gene Expression; Male; Metallothionein; Mice; Mice, Transgenic; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Norepinephrine; Reactive Oxygen Species; Signal Transduction; Stress, Physiological; Transforming Growth Factor beta; Ultrasonography

Cigarette smoking is a devastating risk factor for cardiovascular diseases and nicotine is believed the main toxin component responsible for the toxic myocardial effects of smoking. Nonetheless, neither the precise mechanism of nicotine-induced cardiac dysfunction nor effective treatment is elucidated. The aim of this study was to evaluate the impact of cardiac-specific overexpression of heavy metal scavenger metallothionein on myocardial geometry and mechanical function following nicotine exposure. Adult male friend virus B (FVB) wild-type and metallothionein mice were injected with nicotine (2 mg/kg/d) intraperitoneally for 10 days. Mechanical and intracellular Ca²+ properties were examined. Myocardial histology (cross-sectional area and fibrosis) was evaluated by hematoxylin and eosin (H&E) and Masson trichrome staining, respectively. Oxidative stress and apoptosis were measured by fluoroprobe 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) fluorescence and caspase-3 activity, respectively. Nicotine exposure failed to affect the protein abundance of metallothionein. Our data revealed reduced echocardiographic contractile capacity (fractional shortening), altered cardiomyocyte contractile and intracellular Ca²+ properties including depressed peak shortening amplitude, maximal velocity of shortening/relengthening, resting and electrically-stimulated rise in intracellular Ca²+, as well as prolonged duration of relengthening and intracellular Ca²+ clearance in hearts from nicotine-treated FVB mice, the effect of which was ameliorated by metallothionein. Biochemical and histological findings depicted overt accumulation of reactive oxygen species (ROS), apoptosis and myocardial fibrosis without any change in myocardial cross-sectional area following nicotine treatment, which was mitigated by metallothionein. Taken together, our findings suggest the antioxidant metallothionein may reconcile short-term nicotine exposure-induced myocardial contractile dysfunction and fibrosis possibly through inhibition of ROS accumulation and apoptosis.

Topics: Animals; Calcium; Fibrosis; Heart; Male; Metallothionein; Mice; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Nicotine; Nicotinic Agonists; Reactive Oxygen Species; Smoking

The aim of this study was to investigate the hepatoprotective effects of anthocyanidin delphinidin in carbon tetrachloride (CCl(4))-induced liver fibrosis in mice. Male Balb/C mice were treated with CCl(4) dissolved in olive oil (20%, v/v, 2mL/kg) intraperitoneally (i.p.), twice a week for 7 weeks. Delphinidin was administered i.p. once daily for next 2 weeks, in doses of 10 and 25mg/kg of body weight. The CCl(4) control group has been observed for spontaneous reversion of fibrosis. CCl(4)-administration induced an elevation in serum transaminase and alkaline phosphatase levels and increased oxidative stress in the liver. Delphinidin has successfully attenuated oxidative stress, increased matrix metalloproteinase-9 and metallothionein I/II expression and restored hepatic architecture. Furthermore, the overexpression of tumor necrosis factor-alpha and transforming growth factor-beta1 has been withdrawn by delphinidin. Concomitantly, the expression of alpha-smooth muscle actin indicated returning of hepatic stellate cells (HSC) into inactive state. Our results suggest the therapeutic effects of delphinidin in CCl(4)-induced liver fibrosis by promoting extracellular matrix degradation, HSC inactivation and down-regulation of fibrogenic stimuli, with strong enhancement of hepatic regenerative capability.

Topics: Animals; Anthocyanins; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Down-Regulation; Drug-Related Side Effects and Adverse Reactions; Fibrosis; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Metallothionein; Mice; Mice, Inbred BALB C; Olive Oil; Plant Oils; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

We evaluated metallothionein (MT)-mediated cardioprotection from angiotensin II (Ang II)-induced pathologic remodeling with and without underlying diabetes.. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice are resistant to diabetic cardiomyopathy largely because of the antiapoptotic and antioxidant effects of MT.. The acute and chronic cardiac effects of Ang II were examined in MT-TG and wild-type (WT) mice, and the signaling pathways of Ang II-induced cardiac cell death were examined in neonatal mouse cardiomyocytes.. Acute Ang II administration to WT mice or neonatal cardiomyocytes increased cardiac apoptosis, nitrosative damage, and membrane translocation of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoform p47(phox). These effects were abrogated in MT-TG mice, MT-TG cardiomyocytes, and WT cardiomyocytes pre-incubated with peroxynitrite or superoxide scavengers and NOX inhibitors, suggesting a critical role for NOX activation in Ang II-mediated apoptosis. Prolonged administration of subpressor doses of Ang II (0.5 mg/kg every other day for 2 weeks) also induced apoptosis and nitrosative damage in both diabetic and nondiabetic WT hearts, but not in diabetic and nondiabetic MT-TG hearts. Long-term follow-up (1 to 6 months) of both WT and MT-TG mice after discontinuing Ang II administration revealed progressive myocardial fibrosis, hypertrophy, and dysfunction in WT mice but not in MT-TG mice.. Metallothionein suppresses Ang II-induced NOX-dependent nitrosative damage and cell death in both nondiabetic and diabetic hearts early in the time course of injury and prevents the late development of Ang II-induced cardiomyopathy.

Topics: Angiotensin II; Animals; Apoptosis; Cardiomyopathies; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Fibrosis; Hypertrophy; Metallothionein; Mice; Mice, Transgenic; Myocardium; Myocytes, Cardiac; NADP; Oxidative Stress; Ventricular Remodeling

Zinc is a structural constituent of many proteins, including Cu/Zn superoxide dismutase (SOD), an endogenous antioxidant enzyme. Hypozincemia has been found in patients hospitalized with congestive heart failure, where neurohormonal activation, including the renin-angiotensin-aldosterone system (RAAS), is expected and oxidative stress is present. This study was undertaken to elucidate potential pathophysiological mechanisms involved in Zn dyshomeostasis in aldosteronism. In rats receiving aldosterone/salt treatment (ALDOST) alone for 1 and 4 wk or in combination with spironolactone (Spiro), an ALDO receptor antagonist, we monitored 24-h urinary and fecal Zn excretion and tissue Zn levels in heart, liver, and skeletal muscle, together with tissue metallothionein (MT)-I, a Zn(2+)-binding protein, and Cu/Zn-SOD activities in plasma and tissues. When compared with unoperated, untreated, age-/sex-matched controls, urinary and, in particular, fecal Zn losses were markedly increased (P < 0.05) at days 7 and 28 of ALDOST, leading to hypozincemia and a fall (P < 0.05) in plasma Cu/Zn-SOD activity. Microscopic scars and perivascular fibrosis of intramural coronary arteries first appeared in the right and left ventricles at week 4 of ALDOST and were accompanied by increased (P < 0.05) tissue Zn, MT-I, and Cu/Zn-SOD activity, which were not found in uninjured liver or skeletal muscle. Spiro cotreatment prevented cardiac injury and Zn redistribution to the heart. Thus increased urinary and fecal Zn losses, together with their preferential translocation to sites of cardiac injury, where MT-I overexpression and increased Cu/Zn-SOD activity appeared, contribute to Zn dyshomeostasis in rats with aldosteronism, which were each prevented by Spiro. These findings may shed light on Zn dyshomeostasis found in patients with decompensated heart failure.

Topics: Aldosterone; Animals; Coronary Vessels; Disease Models, Animal; Feces; Fibrosis; Homeostasis; Hyperaldosteronism; Liver; Male; Metabolic Diseases; Metallothionein; Mineralocorticoid Receptor Antagonists; Muscle, Skeletal; Myocardium; Nephrectomy; Oxidative Stress; Rats; Rats, Sprague-Dawley; Spironolactone; Superoxide Dismutase; Time Factors; Zinc

Topics: Animals; Cardiomyopathy, Alcoholic; Disease Models, Animal; Ethanol; Fibrosis; Heart; Metallothionein; Mice; Mice, Knockout; Myocardium; Zinc

Alcohol-induced cardiomyopathy including fibrosis has been recognized clinically for a long time, but its pathogenesis is incompletely understood. Studies using experimental animals have not fully duplicated the pathological changes in humans, and animal models of alcoholic cardiac fibrosis are not available. In the present study, we have developed a mouse model in which cardiac hypertrophy and fibrosis were produced in metallothionein-knockout (MT-KO) mice fed an alcohol-containing liquid diet for 2 months. The same alcohol feeding did not produce cardiac fibrosis in the wild-type (WT) control mice, although there was no difference in the alcohol-induced heart hypertrophy between the WT controls and the MT-KO mice. Zinc supplementation prevented cardiac fibrosis but did not affect heart hypertrophy in the alcohol-fed MT-KO mice, suggesting a specific link between zinc homeostasis and cardiac fibrosis. Serum creatine phosphokinase activity was significantly higher in the alcohol-administered MT-KO mice than in the WT mice, and zinc supplementation decreased serum creatine phosphokinase activities and eliminated the difference between the groups. Thus, disturbance in zinc homeostasis due to the lack of MT associates with alcohol-induced cardiac fibrosis and more severe cardiac injury, making the MT-KO mouse model of alcohol-induced cardiac fibrosis a useful tool to investigate specific factors involved in the alcoholic cardiomyopathy.

Topics: Animals; Cardiomyopathy, Alcoholic; Dietary Supplements; Ethanol; Fibrosis; Heart; Homozygote; Hypertrophy; Liver; Metallothionein; Mice; Mice, Knockout; Myocardium; Zinc

Adult lake whitefish were fed As contaminated diets at nominal concentrations of 0, 1, 10, and 100 microg As/g food (dry weight) for 10, 30, and 64 days. Reduced feed consumption was observed in lake whitefish fed the 100 microg As/g food, beginning on day 45 of exposure. The accumulation and distribution of As in these fish are described in the previous manuscript [Pedlar, R.M., Klaverkamp, J.F., 2001. The accumulation and distribution of dietary arsenic in lake whitefish (Coregonus clupeaformis). Aquat. Toxicol., in press]. At the molecular level of organization, metallothionein (MT) induction occurred in lake whitefish fed the 100 microg As/g food after 10 and 30 days, and in fish fed the 1 and 10 microg As/g diets for 64 days. Dietary As exposure did not have a significant effect on plasma lipid peroxide (LPO) concentrations. At the tissue and organ level, mean liver somatic index decreased significantly in lake whitefish fed the 100 microg As/g food for 64 days. Blood parameters (hematocrit, hemoglobin concentration, red blood cell count, mean cell volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration) were not affected by exposure to As contaminated diets. Liver and gallbladder histopathologies were observed in lake whitefish fed all As contaminated diets after each duration of exposure. Histopathology observed in liver included nuclear, architectural and structural alterations, areas of inflammation, and focal necrosis. Sloughing of the epithelium, dilation of vascular elements, inflammation, edema, fibrosis, and increased width of the submucosa were some of the alterations observed in gallbladders of lake whitefish fed As contaminated diets. Both organs were sensitive to As exposure, as damage occurred with exposure to dietary concentrations of As as low as 1 microg/g. Whole organism parameters were unaltered by dietary As exposure. Based on the results of this study, histopathological alterations in liver and gallbladder, and hepatic MT induction may be useful indicators of As toxicity in environmental monitoring programs that also measure As concentrations in those tissues.

Topics: Administration, Oral; Animal Feed; Animals; Arsenic; Cell Division; Environmental Exposure; Fibrosis; Gallbladder; Inflammation; Lipid Peroxidation; Liver; Metallothionein; Salmonidae; Tissue Distribution; Water Pollutants