Biodistribution Analysis of Peptide-Coated Magnetic Iron Nanoparticles: A Simple and Quantitative Method.

Biodistribution tracks compounds or molecules of interest in vivo to understand a compound's anticipated efficacy and safety. Nanoparticles deliver nucleic acid and drug payloads and enhance tumor permeability due to multiple properties such as high surface area to volume ratio, surface functionalization, and modifications. Studying the in vivo biodistribution of nanoparticles documents the effectiveness and safety of nanoparticles and facilitates a more application-driven approach for nanoparticle development that allows for more successful translation into clinical use. In this study, we present a relatively simple method to determine the biodistribution of magnetic iron nanoparticles in mice. In vitro, cells take up branched amphiphilic peptide-coated magnetic nanobeads (BAPc-MNBs) like their counterparts, i.e., branched amphiphilic peptide capsules (BAPCs) with a hollow water-filled core. Both BAPc-MNBs and BAPCs have widespread applications as a nanodelivery system. We evaluated the BAPc-MNBs tissue distribution in wild-type mice injected intravenously (i.v.), intraperitoneally (i.p.), or orally gavaged to understand the biological interactions and to further the development of branched amphiphilic peptide-based nanoparticles. The magnetic nanoparticles allowed collection of the BAPc-MNBs from multiple organs by magnetic bead sorting, followed by a high-throughput screening for iron content. When injected i.v., nanoparticles were distributed widely to various organs before elimination from the system via the intestines in feces. The spleen accumulated the highest amount of BAPc-MNBs in mice administered NPs via i.v. and i.p. but not via oral gavage. Taken together, these data demonstrate that the magnetic sorting not only allowed quantification of the BAPc-MNBs but also identified the distribution of BAPc-MNBs after distinct administration methods.

Biodistribution Analysis of Peptide-Coated Magnetic Iron Nanoparticles: A Simple and Quantitative Method.

Biodistribution is the tracking of compounds or molecules of interest in the subject which is integral to understanding their anticipated efficacy and safety. Nanoparticles are highly desirable delivery systems which have the ability to deliver higher nucleic acid and drug payloads and they have enhanced tumor permeability due to their unique properties such as high surface area to volume ratio. Studying the biodistribution of nanoparticles is crucial to understand their effectiveness and safety in vivo, facilitate a more application driven approach for nanoparticle development which will lead to their successful translation into clinical use. In this study, we present a relatively simple method to determine the biodistribution of magnetic iron nanoparticles in mice. Branched Amphiphilic Peptide coated Magnetic Nanobeads BAPc-MNBs like their counterpart i.e., Branched Amphiphilic Peptide capsules (BAPCs) with a hollow water-filled core, are readily taken up by cells in vitro and have widespread application as a nanodelivery systems. We evaluated the BAPc-MNBs tissue distribution in wildtype mice injected intravenously (i.v.), intraperitoneally (i.p.) or orally gavaged to understand the biological interactions of the peptide nanoparticles and to further the development of branched amphiphilic peptides-based nanoparticles. BAPc-MNBs were distributed widely to various organs when injected i.v. and were eliminated from the system via the intestines in feces. The spleen was found to accumulate the highest amount of BAPc-MNBs in mice administered the NPs i.v. and i.p. while they were not absorbed into the system via oral gavage. This study not only presents a relatively simple quantification method to determine in vivo biodistribution of magnetic iron nanoparticles that can be widely applied but also demonstrates the potential of Branched Amphiphilic Peptides in the form of BAPCs or BAPc-MNBs as a delivery system.

Toxicity of sodium nitrite-based vertebrate pesticides for European starlings (Sturnus vulgaris).

In the 21st century, invasive animals rank second only to habitat destruction as the greatest threat to global biodiversity. Socially-acceptable and cost-effective strategies are needed to reduce the negative economic and environmental impacts of invasive animals. We investigated the potential for sodium nitrite (SN; CAS 7632-00-0) to serve as an avian toxicant for European starlings (Sturnus vulgaris L.). We also assessed the non-target hazard of an experimental formulation of SN that is being developed as a toxicant for invasive wild pigs (Sus scrofa L.). In gavage experiments with European starlings, we identified a lowest observed adverse effect level (LOAEL) for mortality of 2.40% technical SN (w/v; 120 mg SN/kg body mass) and a no observed adverse effect level (NOAEL) for mortality of 1.30% technical SN (65 mg/kg). The exposure of ten starlings to the experimental formulation of SN (10% SN pig toxicant) resulted in one starling mortality during four days of exposure to the toxic bait. Sodium nitrite toxicity presented a moderate hazard to European starlings; thus, the future development of SN as an avian toxicant is dependent upon its cost-effectiveness. We discuss the management of toxic effects and non-target hazards of SN for wild birds, including best practices for toxic baiting of vertebrate pests and management of invasive wild pigs.

Landscape scale thermoregulatory costs from sublethal exposure to Deep Water Horizon oil in the double-crested cormorant.

Toxic effects of heavy oiling to wildlife are well known from oil spills, although sublethal oil exposure effects are poorly understood. We used Niche Mapper™, to compute spatially and temporally specific energetic and behavioral impacts of repeated sublethal oil exposure to double-crested cormorants (Phalacrocorax auritus). During winter (October-March) cormorants exposed to 13 g, 39 g, and 65-78 g of oil, had on average a 31%, 59%, and 76% predicted increase in total resting energetic requirements (RMR) compared to unoiled birds, respectively. Increased RMR resulted in a mean (±SD) predicted increase in time spent foraging of 36 (±13) min·d-1. During the breeding season (April-September), cormorants had on average a 29%, 57% and 73% increase in total RMR and the mean predicted increase in time spent foraging was 131 (±49) min·d-1. Thermoregulatory effects of sublethal oil exposure may cause greater impacts to bird populations than is currently understood.

Effects of Deepwater Horizon oil on feather structure and thermoregulation in gulls: Does rehabilitation work?

Impacts of large-scale oil spills on avian species are far-reaching. While media attention often focuses on lethal impacts, sub-lethal effects and the impacts of rehabilitation receive less attention. The objective of our study was to characterize effects of moderate external oiling and subsequent rehabilitation on feather structure and thermoregulation in gulls. We captured 30 wild ring-billed gulls (Larus delawarensis) and randomly assigned each individual to an experimental group: 1) controls, 2) rehabilitated birds (externally oiled, rehabilitated by washing), or 3) oiled birds (externally oiled, not rehabilitated). We externally oiled birds with weathered MC252 Deepwater Horizon oil (water for controls) and collected feathers and thermography imagery (FLIR) approximately weekly for four weeks to investigate feather structure (quantified using a barbule clumping index) and thermoregulatory ability (characterized by internal body temperature and external surface temperature). Post-oiling feather clumping was significantly higher in oiled and rehabilitated birds compared to controls, but steadily declined over time in both groups. However, feather microstructure in rehabilitated birds was indistinguishable from controls within three weeks of washing whereas the feathers of oiled birds were still significantly clumped a month post oiling. Internal body temperatures didn't differ in any of the groups, suggesting birds maintain thermoregulatory homeostasis in spite of moderate external oiling. External temperatures for rehabilitated birds didn't differ from controls within a week of rehabilitation. Overall, rehabilitation procedures were effective and washed birds were in better condition compared to non-rehabilitated, oiled birds. This study provides evidence that the benefits of rehabilitation for moderately oiled birds likely outweigh the costs with regard to feather structure and thermoregulation. While feather preening and time were insufficient to reestablish baseline fine scale feather structure in moderately oiled birds, the significant clumping reduction over time may indicate that rehabilitation of lightly oiled birds may not be necessary and deserves further study.

Effects of External Oiling and Rehabilitation on Hematological, Biochemical, and Blood Gas Analytes in Ring-Billed Gulls (Larus delawarensis).

Avian species experience extensive morbidity and mortality following large-scale oil spills, often resulting in oiled birds being rescued, and admitted to rehabilitation. Our objective was to experimentally establish time-specific, descriptive blood analyte data following sublethal oil exposure and subsequent rehabilitation. Thirty wild Ring-billed Gulls (Larus delawarensis) were randomly allocated to three treatment groups of 10 birds each. One treatment group served as controls and two treatment groups were externally oiled daily for 3 days with weathered MC252 oil collected from the Deepwater Horizon oil spill, mimicking the upper threshold of the US Fish and Wildlife Service's moderate oiling classification. Following external oiling, one oiled treatment group was cleaned via standard rehabilitation practices. Serial venous blood samples were collected for a month to measure packed cell volume, total solids, blood gas and select plasma biochemistry analytes, total white blood cell estimates and differentials, and reticulocyte estimates. We found that both sublethal oil exposure and aspects of captivity were associated with a mild non-regenerative anemia. No other differences in venous blood gas and biochemical analytes as well as white blood cell concentrations were observed among the three groups. These findings suggest that the mild anemia seen in oiled birds undergoing rehabilitation is possibly multifactorial and that moderately oiled gulls have subtle, but potentially not insignificant clinicopathological abnormalities following sublethal oil exposure. Oiled gulls did not develop any clinicopathological derangements post-rehabilitation, suggesting current standard practices for rehabilitation cause minimal morbidity in clinically stable, moderately oiled gulls.

Effects of Repeated Sublethal External Exposure to Deep Water Horizon Oil on the Avian Metabolome.

We assessed adverse effects of external sublethal exposure of Deepwater Horizon, Mississippi Canyon 252 oil on plasma and liver metabolome profiles of the double-crested cormorant (Phalacrocorax auritus), a large (1.5 to 3.0 kg) diving waterbird common in the Gulf of Mexico. Metabolomics analysis of avian plasma showed significant negative effects on avian metabolic profiles, in some cases after only two external exposures (26 g cumulative) to oil. We observed significant (p < 0.05) changes in intermediate metabolites of energy metabolism and fatty acid and amino acid metabolic pathways in cormorants after repeated exposure to oil. Exposure to oil increased several metabolites (glycine, betaine, serine and methionine) that are essential to the one-carbon metabolism pathway. Lipid metabolism was affected, causing an increase in production of ketone bodies, suggesting lipids were used as an alternative energy source for energy production in oil exposed birds. In addition, metabolites associated with hepatic bile acid metabolism were affected by oil exposure which was correlated with changes observed in bile acids in exposed birds. These changes at the most basic level of phenotypic expression caused by sublethal exposure to oil can have effects that would be detrimental to reproduction, migration, and survival in avian species.

Experimental and modeled thermoregulatory costs of repeated sublethal oil exposure in the Double-crested Cormorant, Phalacrocorax auritus.

To fully understand the impact of oil exposure, it is important to understand sublethal effects like how increased thermoregulatory costs may affect survival and reproduction. However, it is difficult and time-consuming to measure these effects in wild animals. We present a novel use of a bioenergetics model, Niche Mapper™, to estimate thermoregulatory impacts of oiling, using data from captive Double-crested Cormorants (Phalacrocorax auritus) experimentally exposed to oil. Oiled cormorants had significant increases in surface body temperatures following exposure. Niche Mapper accurately predicted surface temperatures and metabolic rates for unoiled and oiled cormorants and predicted 13-18% increased daily energetic demands due to increased thermoregulatory costs of oiling, consistent with increased food consumption observed in experimentally oiled cormorants. We show that Niche Mapper can provide valuable insight into sublethal oiling effects by quantifying the extent to which thermoregulatory costs divert energy resources away from important life processes like maintenance, reproduction and migration.

Changes in white cell estimates and plasma chemistry measurements following oral or external dosing of double-crested cormorants, Phalacocorax auritus, with artificially weathered MC252 oil.

Scoping studies were designed whereby double-crested cormorants (Phalacocorax auritus) were dosed with artificially weathered Deepwater Horizon (DWH) oil either daily through oil injected feeder fish, or by application of oil directly to feathers every three days. Preening results in oil ingestion, and may be an effective means of orally dosing birds with toxicant to improve our understanding of the full range of physiological effects of oral oil ingestion on birds. Blood samples collected every 5-6 days were analyzed for a number of clinical endpoints including white blood cell (WBC) estimates and differential cell counts. Plasma biochemical evaluations were performed for changes associated with oil toxicity. Oral dosing and application of oil to feathers resulted in clinical signs and statistically significant changes in a number of biochemical endpoints consistent with petroleum exposure. In orally dosed birds there were statistically significant decreases in aspartate amino transferase (AST) and gamma glutamyl transferase (GGT) activities, calcium, chloride, cholesterol, glucose, and total protein concentrations, and increases in plasma urea, uric acid, and phosphorus concentrations. Plasma electrophoresis endpoints (pre-albumin, albumin, alpha-2 globulin, beta globulin, and gamma globulin concentrations and albumin: globulin ratios) were decreased in orally dosed birds. Birds with external oil had increases in urea, creatinine, uric acid, creatine kinase (CK), glutamate dehydrogenase (GLDH), phosphorus, calcium, chloride, potassium, albumin, alpha-1 globulin and alpha-2 globulin. Decreases were observed in AST, beta globulin and glucose. WBC also differed between treatments; however, this was in part driven by monocytosis present in the externally oiled birds prior to oil treatment.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies.

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

Organ weights and histopathology of double-crested cormorants (Phalacrocorax auritus) dosed orally or dermally with artificially weathered Mississippi Canyon 252 crude oil.

A series of toxicity tests were conducted to assess the effects of low to moderate exposure to artificially weathered Deepwater Horizon Mississippi Canyon 252 crude oil on representative avian species as part of the Natural Resource Damage Assessment. The present report summarizes effects of oral exposure (n=26) of double-crested cormorants (DCCO; Phalacrocorax auritus) to 5 or 10ml oil kg-1 day-1 for up to 21 days or dermal application (n=25) of 13ml oil to breast and back feathers every three days totaling 6 applications in 21 days on organ weights and histopathology. Absolute and relative kidney and liver weights were increased in birds exposed to oil. Additionally, gross and/or histopathologic lesions occurred in the kidney, heart, pancreas and thyroid. Clinically significant renal lesions in the orally dosed birds included squamous metaplasia and increased epithelial hypertrophy of the collecting ducts and renal tubules and mineralization in comparison to controls. Gross cardiac lesions including thin walls and flaccid musculature were documented in both orally and dermally dosed birds and myocardial fibrosis was found in low numbers of dermally dosed birds only. Cytoplasmic vacuolation of the exocrine pancreas was noted in orally dosed birds only. Thyroid follicular hyperplasia was increased in dermally dosed birds only possibly due to increased metabolism required to compensate damaged feather integrity and thermoregulate. Gastrointestinal ulceration was found in orally dosed birds only. There were no significant hepatic histopathologic lesions induced by either exposure route. Therefore, hepatic histopathology is likely not a good representation of oil-induced damage. Taken together, the results suggest that oral or dermal exposure of DCCOs to artificially weathered MC252 crude oil induced organ damage that could potentially affect survivability.

Dietary intake of Deepwater Horizon oil-injected live food fish by double-crested cormorants resulted in oxidative stress.

The Deepwater Horizon oil spill released 134 million gallons of crude oil into the Gulf of Mexico making it the largest oil spill in US history and exposing fish, birds, and marine mammals throughout the Gulf of Mexico to its toxicity. Fish eating waterbirds such as the double-crested cormorant (Phalacrocorax auritus) were exposed to the oil both by direct contact with the oil and orally through preening and the ingestion of contaminated fish. This study investigated the effects of orally ingestedMC252 oil-contaminated live fish food by double-crested cormorants on oxidative stress. Total, reduced, and oxidized glutathione levels, superoxide dismutase and glutathione peroxidase activities, total antioxidant capacity and lipid peroxidation were assessed in the liver tissues of control and treated cormorants. The results suggest that ingestion of the oil-contaminated fish resulted in significant increase in oxidative stress in the liver tissues of these birds. The oil-induced increase in oxidative stress could have detrimental impacts on the bird's life-history.

Reprint of: Development of methods for avian oil toxicity studies using the double crested cormorant (Phalacrocorax auritus).

Oral and external dosing methods replicating field exposure were developed using the double crested cormorant (DCCO) to test the toxicity of artificially weathered Deepwater Horizon Mississippi Canyon 252 oil. The majority of previous oil dosing studies conducted on wild-caught birds used gavage methods to dose birds with oil and determine toxicity. However, rapid gut transit time of gavaged oil likely reduces oil absorption. In the present studies, dosing relied on injection of oil into live feeder fish for oral dosing of these piscivorous birds, or applying oil to body contour feathers resulting in transdermal oil exposure and oral exposure through preening. Both oral and external oil dosing studies identified oil-related toxicity endpoints associated with oxidative stress such as hemolytic anemia, liver and kidney damage, and immuno-modulation or compromise. External oil application allowed for controlled study of thermoregulatory stress as well. Infrared thermal images indicated significantly greater surface temperatures and heat loss in treated birds following external oil applications; however, measurements collected by coelomically implanted temperature transmitters showed that internal body temperatures were stable over the course of the study period. Birds exposed to oil externally consumed more fish than control birds, indicating metabolic compensation for thermal stress. Conversely, birds orally dosed with oil experienced hypothermia and consumed less fish compared to control birds.

Reprint of: CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil.

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to deepwater Horizon Mississippi Canyon 252 oil.

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5ml/kg bw oil or 10ml/kg bw oil, 21day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Development of methods for avian oil toxicity studies using the double crested cormorant (Phalacrocorax auritus).

Oral and external dosing methods replicating field exposure were developed using the double crested cormorant (DCCO) to test the toxicity of artificially weathered Deepwater Horizon Mississippi Canyon 252 oil. The majority of previous oil dosing studies conducted on wild-caught birds used gavage methods to dose birds with oil and determine toxicity. However, rapid gut transit time of gavaged oil likely reduces oil absorption. In the present studies, dosing relied on injection of oil into live feeder fish for oral dosing of these piscivorous birds, or applying oil to body contour feathers resulting in transdermal oil exposure and oral exposure through preening. Both oral and external oil dosing studies identified oil-related toxicity endpoints associated with oxidative stress such as hemolytic anemia, liver and kidney damage, and immuno-modulation or compromise. External oil application allowed for controlled study of thermoregulatory stress as well. Infrared thermal images indicated significantly greater surface temperatures and heat loss in treated birds following external oil applications; however, measurements collected by coelomically implanted temperature transmitters showed that internal body temperatures were stable over the course of the study period. Birds exposed to oil externally consumed more fish than control birds, indicating metabolic compensation for thermal stress. Conversely, birds orally dosed with oil experienced hypothermia and consumed less fish compared to control birds.

Effect of angiotensin II on primary cardiac fibroblast matrix metalloproteinase activities.

Left ventricular dysfunction is associated with reperfusion injury occurring during open-heart surgery. There is an increased secretion of angiotensin II (Ang II) and increased matrix metalloproteinases (MMPs) activities associated with open-heart surgery that may affect the cardiac extracellular matrix (ECM). The goal of this study was to determine the effects of Ang II and selective angiotensin II receptor (AT1-R and AT2-R) blockers on the enzymatic activities of MMPs in primary adult murine cardiac fibroblasts (CF). Our hypothesis is that Aug II, with and without a selective receptor blocker, differentially affects CF MMPs activities. The CF were treated with Ang II (10(-6) M) and doses of AT1-R and AT2-R blockers (losartan and PD123319, respectively) at doses of 10(-7) to 10(-5) M for 48 hours. The Ang II-stimulated CF reduced collagenase activities by only 24% (p = 0.004); however, the MMP-2 and MMP-9 gelatinase activities were reduced by 42% and 39%, respectively (p = 0.022). The losartan dose dependently increased MMP-2 (p = 0.02) and MMP-9 (ns). PD123319 at 10(-5) M significantly reduced MMP-2 and MMP-9 activities compared with the Ang II group (p = 0.014 and p = 0.02, respectively). The doses of PD123319 at 10(-6) and 10(-7) M increased the MMP-2 and MMP-9 enzymatic activities significantly above the Ang II only group. Thus, Ang II and AT1-R and AT2-R differentially affect the collagenase and gelatinase MMPs activities released by cardiac fibroblasts.

Relationship of aging and cardiac IL-10.

Current therapies for the treatment of myocardial infarction and heart failure include medical, surgical, mechanical assist, and transplantation. These therapies have been based on the dogma that ventricular myocytes themselves are terminally differentiated and, therefore, cannot regenerate. This concept has been recently challenged with stem cell therapy. A potential problem is the ability of cardiac tissue to mobilize, recruit, and transdifferentiate adult stem cells from other tissues. We believe that there is a unique failure of the damaged myocardium to provide the appropriate molecular signals for stem cells engraftment related to age. Our hypothesis is that the overexpression of IL-10 in the aged population reduces cardiac cellular proliferation subsequent to myocardial injury. This hypothesis is supported by aging models, where elevated levels of IL-10 are associated with reduced healing response to noncardiac tissue injury. We demonstrated an increased cardiac gene expression of IL-10 that may be associated with a reduced proliferative response in the border regions of the infarcted myocardium that are proportional with age. In conclusion, myocardial infarction and heart failure has presented a significant challenge for the clinician to provide reparative therapies. The use of therapeutics to modulate IL-10 and, thereby, optimizing regenerative processes in the injured myocardium may provide a unique means for the cardiac patient.