Pulmonary pathogenesis in a murine model of inhaled arsenical exposure.

Arsenic trioxide (ATO), an inorganic arsenical, is a toxic environmental contaminant. It is also a widely used chemical with industrial and medicinal uses. Significant public health risk exists from its intentional or accidental exposure. The pulmonary pathology of acute high dose exposure is not well defined. We developed and characterized a murine model of a single inhaled exposure to ATO, which was evaluated 24 h post-exposure. ATO caused hypoxemia as demonstrated by arterial blood-gas measurements. ATO administration caused disruption of alveolar-capillary membrane as shown by increase in total protein and IgM in the bronchoalveolar lavage fluid (BALF) supernatant and an onset of pulmonary edema. BALF of ATO-exposed mice had increased HMGB1, a damage-associated molecular pattern (DAMP) molecule, and differential cell counts revealed increased neutrophils. BALF supernatant also showed an increase in protein levels of eotaxin/CCL-11 and MCP-3/CCL-7 and a reduction in IL-10, IL-19, IFN-γ, and IL-2. In the lung of ATO-exposed mice, increased protein levels of G-CSF, CXCL-5, and CCL-11 were noted. Increased mRNA levels of TNF-a, and CCL2 in ATO-challenged lungs further supported an inflammatory pathogenesis. Neutrophils were increased in the blood of ATO-exposed animals. Pulmonary function was also evaluated using flexiVent. Consistent with an acute lung injury phenotype, respiratory and lung elastance showed significant increase in ATO-exposed mice. PV loops showed a downward shift and a decrease in inspiratory capacity in the ATO mice. Flow-volume curves showed a decrease in FEV0.1 and FEF50. These results demonstrate that inhaled ATO leads to pulmonary damage and characteristic dysfunctions resembling ARDS in humans.

Chronic cardiac structural damage, diastolic and systolic dysfunction following acute myocardial injury due to bromine exposure in rats.

Accidental bromine spills are common and its large industrial stores risk potential terrorist attacks. The mechanisms of bromine toxicity and effective therapeutic strategies are unknown. Our studies demonstrate that inhaled bromine causes deleterious cardiac manifestations. In this manuscript we describe mechanisms of delayed cardiac effects in the survivors of a single bromine exposure. Rats were exposed to bromine (600 ppm for 45 min) and the survivors were sacrificed at 14 or 28 days. Echocardiography, hemodynamic analysis, histology, transmission electron microscopy (TEM) and biochemical analysis of cardiac tissue were performed to assess functional, structural and molecular effects. Increases in right ventricular (RV) and left ventricular (LV) end-diastolic pressure and LV end-diastolic wall stress with increased LV fibrosis were observed. TEM images demonstrated myofibrillar loss, cytoskeletal breakdown and mitochondrial damage at both time points. Increases in cardiac troponin I (cTnI) and N-terminal pro brain natriuretic peptide (NT-proBNP) reflected myofibrillar damage and increased LV wall stress. LV shortening decreased as a function of increasing LV end-systolic wall stress and was accompanied by increased sarcoendoplasmic reticulum calcium ATPase (SERCA) inactivation and a striking dephosphorylation of phospholamban. NADPH oxidase 2 and protein phosphatase 1 were also increased. Increased circulating eosinophils and myocardial 4-hydroxynonenal content suggested increased oxidative stress as a key contributing factor to these effects. Thus, a continuous oxidative stress-induced chronic myocardial damage along with phospholamban dephosphorylation are critical for bromine-induced chronic cardiac dysfunction. These findings in our preclinical model will educate clinicians and public health personnel and provide important endpoints to evaluate therapies.

Acute pulmonary effects of aerosolized nicotine.

Nicotine is a highly addictive principal component of both tobacco and electronic cigarette that is readily absorbed in blood. Nicotine-containing electronic cigarettes are promoted as a safe alternative to cigarette smoking. However, the isolated effects of inhaled nicotine are largely unknown. Here we report a novel rat model of aerosolized nicotine with a particle size (~1 µm) in the respirable diameter range. Acute nicotine inhalation caused increased pulmonary edema and lung injury as measured by enhanced bronchoalveolar lavage fluid protein, IgM, lung wet-to-dry weight ratio, and high-mobility group box 1 (HMGB1) protein and decreased lung E-cadherin protein. Immunohistochemical analysis revealed congested blood vessels and increased neutrophil infiltration. Lung myeloperoxidase mRNA and protein increased in the nicotine-exposed rats. Complete blood counts also showed an increase in neutrophils, white blood cells, eosinophils, and basophils. Arterial blood gas measurements showed an increase in lactate. Lungs of nicotine-inhaling animals revealed increased mRNA levels of IL-1A and CXCL1. There was also an increase in IL-1α protein. In in vitro air-liquid interface cultures of airway epithelial cells, there was a dose dependent increase in HMGB1 release with nicotine treatment. Air-liquid cultures exposed to nicotine also resulted in a dose-dependent loss of barrier as measured by transepithelial electrical resistance and a decrease in E-cadherin expression. Nicotine also caused a dose-dependent increase in epithelial cell death and an increase in caspase-3/7 activities. These results show that the nicotine content of electronic cigarettes may have adverse pulmonary and systemic effects.

Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats.

Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.

Desmin loss and mitochondrial damage precede left ventricular systolic failure in volume overload heart failure.

Heart failure due to chronic volume overload (VO) in rats and humans is characterized by disorganization of the cardiomyocyte desmin/mitochondrial network. Here, we tested the hypothesis that desmin breakdown is an early and continuous process throughout VO. Male Sprague-Dawley rats had aortocaval fistula (ACF) or sham surgery and were examined 24 h and 4 and 12 wk later. Desmin/mitochondrial ultrastructure was examined by transmission electron microscopy (TEM) and immunohistochemistry (IHC). Protein and kinome analysis were performed in isolated cardiomyocytes, and desmin cleavage was assessed by mass spectrometry in left ventricular (LV) tissue. Echocardiography demonstrated a 40% decrease in the LV mass-to-volume ratio with spherical remodeling at 4 wk with ACF and LV systolic dysfunction at 12 wk. Starting at 24 h and continuing to 4 and 12 wk, with ACF there is TEM evidence of extensive mitochondrial clustering, IHC evidence of disorganization associated with desmin breakdown, and desmin protein cleavage verified by Western blot analysis and mass spectrometry. IHC results revealed that ACF cardiomyocytes at 4 and 12 wk had perinuclear translocation of αB-crystallin from the Z disk with increased α, ß-unsaturated aldehyde 4-hydroxynonelal. Use of protein markers with verification by TUNEL staining and kinome analysis revealed an absence of cardiomyocyte apoptosis at 4 and 12 wk of ACF. Significant increases in protein indicators of mitophagy were countered by a sixfold increase in p62/sequestosome-1, which is indicative of an inability to complete autophagy. An early and continuous disruption of the desmin/mitochondrial architecture, accompanied by oxidative stress and inhibition of apoptosis and mitophagy, suggests its causal role in LV dilatation and systolic dysfunction in VO.NEW & NOTEWORTHY This study provides new evidence of early onset (24 h) and continuous (4-12 wk) desmin misarrangement and disruption of the normal sarcomeric and mitochondrial architecture throughout the progression of volume overload heart failure, suggesting a causal link between desmin cleavage and mitochondrial disorganization and damage.

Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload.

BACKGROUND: Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. METHODS AND RESULTS: Isolated LV fibroblasts taken from 4 and 12week ACF Sprague-Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase's role in autophagy induction. CONCLUSION: In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation.

Volume overload induces autophagic degradation of procollagen in cardiac fibroblasts.

In a pure volume overloaded (VO) heart, interstitial collagen loss is degraded by matrix metalloproteinases (MMPs) that leads to left ventricular (LV) dilatation and heart failure. Cardiac fibroblasts are the primary source of extracellular matrix proteins that connect cardiomyocytes. The goal of this study was to determine how VO affects intracellular procollagen in cardiac fibroblasts. Using the aortocaval fistula (ACF) model in Sprague-Dawley rats, we demonstrate that cardiac fibroblasts isolated from 4 and 12 wk ACF animals have decreased intracellular procollagen I compared to the fibroblasts from age-matched shams. The reduction of procollagen I is associated with increased autophagy as demonstrated by increased autophagic vacuoles and LC3-II expression. To test the relationship between autophagy and procollagen degradation, we treated adult cardiac fibroblasts with either an autophagy inducer, rapamycin, or an inhibitor, wortmannin, and found that procollagen I protein levels were decreased in fibroblasts treated with rapamycin and elevated in wortmannin-treated cells. In addition, we demonstrated that VO induces oxidative stresses in cardiac fibroblasts from 4 and 12 wk ACF rats. Treatment of cultured cardiac fibroblasts with an oxidative stress-inducing agent (DMNQ) induces autophagy and intracellular procollagen I and fibronectin degradation, which is reversed by wortmannin but not by the global MMP inhibitor (PD166793). Mechanical stretch of cardiac fibroblasts also induces oxidative stress and autophagic degradation of procollagen I and fibronectin. Our results suggest that in addition to the well-known effects of MMPs on extracellular collagen degradation in VO, there is a concurrent degradation of intracellular procollagen and fibronectin mediated by oxidative stress-induced autophagy in cardiac fibroblasts.

Mitochondrial genetic background modulates bioenergetics and susceptibility to acute cardiac volume overload.

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial-nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the 'mitochondrial paradigm' for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress.

Chymase inhibition prevents fibronectin and myofibrillar loss and improves cardiomyocyte function and LV torsion angle in dogs with isolated mitral regurgitation.

BACKGROUND: The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. METHODS AND RESULTS: We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a >40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P<0.05) and normalized in MR+CI dogs (4.85±0.44%). MRI with tissue tagging demonstrated an increase in LV torsion angle in MR+CI versus MR dogs. CI normalized the significant decrease in fibronectin and FAK phosphorylation and prevented cardiomyocyte myofibrillar degeneration in MR dogs. In addition, total titin and its stiffer isoform were increased in the LV epicardium and paralleled the changes in fibronectin and FAK phosphorylation in MR+CI dogs. CONCLUSIONS: These results suggest that chymase disrupts cell surface-fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR.

Dynamic molecular and histopathological changes in the extracellular matrix and inflammation in the transition to heart failure in isolated volume overload.

Left ventricular (LV) volume overload (VO) causes eccentric remodeling with inflammatory cell infiltration and extracellular matrix (ECM) degradation, for which there is currently no proven therapy. To uncover new pathways that connect inflammation and ECM homeostasis with cellular dysfunction, we determined the cardiac transciptome in subacute, compensated, and decompensated stages based on in vivo hemodynamics and echocardiography in the rat with aortocaval fistula (ACF). LV dilatation at 5 wk was associated with a normal LV end-diastolic dimension-to-posterior wall thickness ratio (LVEDD/PWT; compensated), whereas the early 2-wk (subacute) and late 15-wk (decompensated) ACF groups had significant increases in LVEDD/PWT. Subacute and decompensated stages had a significant upregulation of genes related to inflammation, the ECM, the cell cycle, and apoptosis. These changes were accompanied by neutrophil and macrophage infiltration, nonmyocyte apoptosis, and interstitial collagen loss. At 15 wk, there was a 40-fold increase in the matricellular protein periostin, which inhibits connections between collagen and cells, thereby potentially mediating a side-to-side slippage of cardiomyocytes and LV dilatation. The majority of downregulated genes was composed of mitochondrial enzymes whose suppression progressed from 5 to 15 wk concomitant with LV dilatation and systolic heart failure. The profound decrease in gene expression related to fatty acid, amino acid, and glucose metabolism was associated with the downregulation of peroxisome proliferator associated receptor (PPAR)-α-related and bioenergetic-related genes at 15 wk. In VO, an early phase of inflammation subsides at 5 wk but reappears at 15 wk with marked periostin production along with the suppression of genes related to PPAR-α and energy metabolism.

Cutaneous lewisite exposure causes acute lung injury.

Lewisite is a strong vesicating and chemical warfare agent. Because of the rapid transdermal absorption, cutaneous exposure to lewisite can also elicit severe systemic injury. Lewisite (2.5, 5.0, and 7.5 mg/kg) was applied to the skin of Ptch1+/- /SKH-1 mice and acute lung injury (ALI) was assessed after 24 hours. Arterial blood gas measurements showed hypercapnia and hypoxemia in the lewisite-exposed group. Histological evaluation of lung tissue revealed increased levels of proinflammatory neutrophils and a dose-dependent increase in structural changes indicative of injury. Increased inflammation was also confirmed by altered expression of cytokines, including increased IL-33, and a dose-dependent elevation of CXCL1, CXCL5, and GCSF was observed in the lung tissue. In the bronchoalveolar lavage fluid of lewisite-exposed animals, there was a significant increase in HMGB1, a damage-associated molecular pattern molecule, as well as elevated CXCL1 and CXCL5, which coincided with an influx of neutrophils to the lungs. Complete blood cell analysis revealed eosinophilia and altered neutrophil-lymphocyte ratios as a consequence of lewisite exposure. Mean platelet volume and RBC distribution width, which are predictors of lung injury, were also increased in the lewisite group. These data demonstrate that cutaneous lewisite exposure causes ALI and may contribute to mortality in exposed populations.

Apocynin attenuates tubular apoptosis and tubulointerstitial fibrosis in transgenic mice independent of hypertension.

Angiotensin II stimulates the formation of reactive oxygen species by increased NADPH oxidase activity, which contributes to proapoptotic and profibrotic mechanisms critical in renal injury. Here we determine if apocynin, an inhibitor of NADPH oxidase, interferes with the action of the intrarenal renin-angiotensin system to minimize the progression of renal disease. Transgenic mice that overexpress rat angiotensinogen in their proximal tubule cells were given either apocynin, perindopril, or hydralazine while untreated or apocynin-treated non-transgenic littermates served as controls. Untreated transgenic mice had significant elevations of their systolic blood pressure, albuminuria, reactive oxygen species production, NADPH oxidase activity, tubular apoptosis, active caspase-3, Bax, transforming growth factor-beta1, plasminogen activator inhibitor-1, extracellular matrix proteins, collagen type IV, and phosphorylated p47phox expression compared to untreated non-transgenic mice. Apocynin and perindopril blunted these changes; however, apocynin had no effect on the systolic blood pressure whereas hydralazine prevented hypertension and tubulointerstitial fibrosis but not proximal tubule cell apoptosis. Our study shows that the intrarenal renin-angiotensin system stimulates proximal tubule cell apoptosis and tubulointerstitial fibrosis, in part, by enhanced NADPH oxidase activity and reactive oxygen species generation independent of systemic hypertension.

Overexpression of angiotensinogen increases tubular apoptosis in diabetes.

The intrarenal renin-angiotensin system (RAS) plays an important role in the progression of diabetic nephropathy. We have previously reported that mice overexpressing angiotensinogen in renal proximal tubular cells (RPTC) develop hypertension, albuminuria, and renal injury. Here, we investigated whether activation of the intrarenal RAS contributes to apoptosis of RPTC in diabetes. Induction of diabetes with streptozotocin in these transgenic mice led to significant increases in BP, albuminuria, RPTC apoptosis, and proapoptotic gene expression compared with diabetic nontransgenic littermates. Insulin and/or RAS blockers markedly attenuated these changes. Hydralazine prevented hypertension but not albuminuria, RPTC apoptosis, or proapoptotic gene expression. In vitro, high-glucose medium significantly increased apoptosis and caspase-3 activity in rat immortalized RPTC overexpressing angiotensinogen compared with control cells, and these changes were prevented by insulin and/or RAS blockers. In conclusion, intrarenal RAS activation and high glucose may act in concert to increase tubular apoptosis in diabetes, independent of systemic hypertension.

Chymase uptake by cardiomyocytes results in myosin degradation in cardiac volume overload.

BACKGROUND: Volume overload (VO) of isolated mitral regurgitation (MR) or aortocaval fistula (ACF) is associated with extracellular matrix degradation and cardiomyocyte myofibrillar and desmin breakdown. Left ventricular (LV) chymase activity is increased in VO and recent studies demonstrate chymase presence within cardiomyocytes. Here we test the hypothesis that chymase within the cardiomyocyte coincides with myosin and desmin breakdown in VO. METHODS AND RESULTS: Aortocaval fistula (ACF) was induced in Sprague Dawley (SD) rats and was compared to age-matched sham-operated rats at 24 hours, 4 and 12 weeks. Immunohistochemistry (IHC) and transmission electron microscopy (TEM) immunogold of LV tissue demonstrate chymase within cardiomyocytes at all ACF time points. IHC for myosin demonstrates myofibrillar disorganization starting at 24 hours. Proteolytic presence of chymase in cardiomyocytes is verified by in situ chymotryptic tissue activity that is inhibited by pretreatment with a chymase inhibitor. Real-time PCR of isolated cardiomyocytes at all ACF time points and in situ hybridization demonstrate endothelial cells and fibroblasts as a major source of chymase mRNA in addition to mast cells. Chymase added to adult rat cardiomyocytes in vitro is taken up by a dynamin-mediated process and myosin breakdown is attenuated by dynamin inhibitor, suggesting that chymase uptake is essential for myosin breakdown. In a previous study in the dog model of chronic MR, the intracellular changes were attributed to extracellular effects. However, we now demonstrate intracellular effects of chymase in both species. CONCLUSION: In response to VO, fibroblast and endothelial cells produce chymase and subsequent cardiomyocyte chymase uptake is followed by myosin degradation. The results demonstrate a novel intracellular chymase-mediated mechanism of cardiomyocyte dysfunction and adverse remodeling in a pure VO.

Novel TLR7 agonist stimulates activity of CIK/NK immunological effector cells to enhance antitumor cytotoxicity.

Toll-like receptor (TLR) 7/8 agonists have been applied in combination with chemo-, radio- or immunotherapy for lymphoma, and used as topical drugs for the treatment of viral skin lesions and skin tumors. In the present study, the role of an adenine analog, 9-(4-carboxyphenyl)-8-hydroxy-2-(2-methoxyethoxy)-adenine [termed Gao Dong (GD)], a novel TLR7 agonist, in the activation of cytokine-induced killer/natural killer (CIK/NK) cells was determined. The results of the present study indicated that GD was able to activate CIK/NK cells. The proportion of GD-induced CD3+CD56+ CIK and CD3-CD56+ NK cells was ~4% higher respectively compared with the control. Notably, combination therapy with CIK/NK cells stimulated by GD, markedly suppressed the proliferation of the chronic myelogenous leukemia K562 cell line. Following GD treatment, the cytotoxicity improved by ~25 and 21% when the effector/target ratio was 20:1 and 10:1, respectively. The results of the present study suggested a novel protocol for CIK/NK cell proliferation and revealed that GD may serve as a potent innate and adaptive immunomodulator in immunocyte culture.

Neuroprotection of Transplanting Human Umbilical Cord Mesenchymal Stem Cells in a Microbead Induced Ocular Hypertension Rat Model.

PURPOSE: The purpose of this study is to investigate the potential therapeutic benefits of intravitreally transplanted human umbilical cord mesenchymal stem cells (UC-MSCs) in an animal model of microbead-injection-induced ocular hypertension (OHT). METHODS: UC-MSCs were isolated from human umbilical cords and then cultured. The OHT model was induced via intracameral injection of polystyrene microbeads in Sprague-Dawley adult rat eyes. Fifty-four healthy adult rats were randomly divided into three groups: normal control, OHT model treated with intravitreal transplantation of UC-MSCs, or phosphate-buffered saline (PBS). Two days after OHT was induced, either 5 µl 105 UC-MSCs suspension or PBS was injected into the vitreous cavity of rats. UC-MSCs localization and integration were examined via immunohistochemistry. Neuroprotection was quantified by counting retinal ganglion cells (RGCs) and axons 2 weeks following transplantation. The expression levels of glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and glial fibrillary acidic protein (GFAP) were assessed via immunohistochemistry and Western blot. Functional recovery was assessed 2 weeks after transplantation via scotopic threshold response (STR) electroretinography. RESULTS: Elevated IOP levels were sustained at least 3 weeks after intracameral microbead injection and the number of ß-III-tubulin+ RGCs significantly declined compared to PBS-injected eyes. UC-MSCs survived for at least 2 weeks after intravitreal transplantation and predominantly located in the vitreous cavity. A fraction of cells migrated into the ganglion cell layer of host retina, but without differentiation. Intravitreal UC-MSC transplantation resulted in increased number of RGCs, axons, and increased expression of GDNF and BDNF but decreased expression of GFAP. Intravitreal delivery of UC-MSCs significantly improved the recovery of the positive STR. CONCLUSIONS: Intravitreal transplantation of UC-MSCs revealed the neuroprotection in the microbead-injection induced OHT. The effects could be related to the secretion of tropic factors (BDNF and GDNF) and the modulation of glial cell activation.

Synergy of purine-scaffold TLR7 agonist with doxorubicin on systemic inhibition of lymphoma in mouse model.

Chemo- and radio-therapy suffer from certain well-recognized drawbacks for lymphoma therapy. Passive immunotherapy with monoclonal antibody has improved outcome for patients with CD20+ B cell lymphoma, but not for T cell lymphoma. Therefore, novel treatment approaches are clearly required for T cell lymphoma. To date, the combined application targeting TLR7, 8 and 9 has established long-term antitumor immunity. We previously synthesized a purine-scaffold TLR7 agonist named GD5. Here, we report that the intratumoral administration of GD5 combined with doxorubicin (DOX), a conventional chemotherapeutic agent in T cell lymphoma. This combined treatment made mice to produce more cytokines in blood, and generate more potent cytotoxic T lymphocyte response, then result in effective eradication of both local and distant tumors in tumor-bearing mice. Our findings demonstrate the potential for enhancing the efficacy of the current standard DOX therapy through combination with TLR7 agonist GD5 to improve antitumor immune responses and provide durable remissions for T cell lymphoma.

Short-acting beta-adrenergic antagonist esmolol given at reperfusion improves survival after prolonged ventricular fibrillation.

BACKGROUND: High catecholamine concentrations are cytotoxic to cardiac myocytes. We hypothesized that myocardial interstitial catecholamine levels are greatly elevated immediately after long-duration ventricular fibrillation (VF), defibrillation, and reperfusion and that the short-acting beta-antagonist esmolol administered at reperfusion would protect against this catecholamine surge and improve survival. METHODS AND RESULTS: In part 1 of this study, catecholamines from myocardial interstitial fluid (ISF) and aortic and coronary sinus plasma were quantified by use of 3H-labeled radioenzymatic assay in 8 open-chest, anesthetized pigs. Eight minutes of electrically induced VF was followed by internal defibrillation and reperfusion. By 4 minutes of VF, ISF norepinephrine increased significantly, from 1.3+/-0.3 to 7.4+/-2.4 ng/mL. Epinephrine increased significantly, from 0.4+/-0.2 to 1.5+/-0.7 ng/mL. ISF norepinephrine and epinephrine peaked at 219.2+/-92.1 and 63.7+/-25.1 ng/mL after defibrillation and reperfusion and decreased significantly to 12.2+/-3.5 and 6.7+/-3.1 ng/mL 23 minutes after defibrillation. Transcardiac catecholamine changes were similar. In part 2, 8 minutes of VF was followed by external defibrillation in anesthetized, closed-chest pigs. Animals received 1.0 mg/kg esmolol (n=8) or saline (n=8) intravenously at the start of cardiopulmonary resuscitation (CPR). Advanced cardiac life support, including CPR and epinephrine, was delivered to both groups. Esmolol before reperfusion improved return of spontaneous circulation and 4-hour survival (7/8 versus 3/8 survivors, chi2 P<0.05). CONCLUSIONS: Transcardiac and ISF norepinephrine and epinephrine levels are briefly massively elevated after 8 minutes of VF, defibrillation, and reperfusion. A short-acting beta-antagonist administered immediately after defibrillation improves return of spontaneous circulation and 4-hour survival after this prolonged VF.

Beta1-adrenergic receptor blockade attenuates angiotensin II-mediated catecholamine release into the cardiac interstitium in mitral regurgitation.

BACKGROUND: This study tested the hypothesis that beta1-adrenoreceptor blockade modulates the angiotensin II (Ang II)-evoked neural release of norepinephrine (NE) and epinephrine (Epi) into the cardiac interstitial fluid (ISF) space in experimentally induced mitral regurgitation (MR) in the dog. METHODS AND RESULTS: Normal dogs (n=8) were compared with dogs with MR of 2 (n=8) and 4 (n=6) weeks' duration and with dogs with MR treated with beta1-receptor blockade (RB; extended-release metoprolol succinate, 100 mg QD; MR+beta1-RB) that was started 24 hours after MR induction for 2 (n=6) and 4 weeks (n=8). Left ventricular end-diastolic dimension increased 20% as plasma Ang II levels increased >5-fold in both MR and MR+beta1-RB dogs at 2 and 4 weeks. Ang II infusion into the left atrium produced increases in ISF NE and Epi in normal dogs, which were further increased in 2- and 4-week MR dogs but were restored to normal in 4-week MR+beta1-RB dogs. Ang II infusion produced 4-fold increases in circulating NE and Epi in 2- and 4-week MR dogs that returned to normal in 4-week+beta1-RB dogs. Left ventricular angiotensin-converting enzyme activity and ISF Ang II were increased in 4-week MR dogs but were decreased in 4-week MR+beta1-RB dogs. CONCLUSIONS: beta1-RB decreases renin-angiotensin system sympathostimulation and activation by attenuating the Ang II-mediated NE and Epi release into the cardiac ISF and circulation and by decreasing left ventricular angiotensin-converting enzyme expression in the early phases of volume overload.