The role of GILZ in modulation of adaptive immunity in a murine model of myocardial infarction.

Myocardial infarction (MI) is associated with intense immune and inflammatory responses which contribute to tissue injury. Increasing evidence indicates that the glucocorticoid-induced leucine zipper (GILZ) protein suppresses immune and inflammatory responses. However, the status of and the role of GILZ in MI are not known. We tested the hypotheses that a) MI reduces cardiac GILZ associated with intense inflammation and cell death and b) intramyocardial GILZ delivery confers cardioprotection in association with increased Tregs and suppression of inflammation. Male Balb/C mice were subjected to MI or sham operation; the infarcted animals were subdivided to receive intramyocardial injections of PBS, GILZ overexpressing cells (GILZ) or their controls expressing the green fluorescent protein (GFP). Three hours after the procedures, hearts were procured for subsequent analyses. MI markedly reduced cardiac GILZ expression accompanied with a) increase in Th-17 cells (i.e., CD3+CD4+IL-17+ BNP-) but decrease in Tregs (i.e., CD3+CD4+FoxP3+BNP-), and b) disruption of mitochondrial membrane potential (ψm) associated with significant increases in apoptotic and necrotic cell death. While both GILZ and GFP returned the aforementioned parameters towards those of sham controls, these effects were most marked for mice receiving GILZ. Thus, GILZ markedly reduced Th-17 cells but increased Tregs and the anti-inflammatory cytokine, IL-10 positive cells accompanied with preservation of ψm and prevention of cell death. To our knowledge, this is the first report indicating an important role for GILZ in MI, in part via modulation of adaptive immune response, which raises the prospect of exogenous GILZ delivery as a novel cardioprotective modality.

Endoplasmic reticulum stress response and inflammatory cytokines in type 2 diabetic nephropathy: role of indoleamine 2,3-dioxygenase and programmed death-1.

We tested the hypotheses that a) type 2 diabetes increases endoplasmic reticulum (ER) stress response, production of pro-inflammatory cytokines and kidney cell death and b) downregulations of renal indoleamine 2,3-dioxygenase (IDO) and programmed death-1 (PD-1) contribute to exacerbated inflammation and tissue injury. The growth arrest and DNA damage-inducible protein 153 (GADD153; a marker of ER stress response), inflammatory cytokines and cell death were determined in the context of assessment of IDO and PD-1 in an animal model of type 2 diabetic nephropathy (i.e., db/db mouse). Peripheral blood of 4-month-old db/db mice manifested significantly greater percents of interleukin (IL)-17 and IL-23 positive cells in association with greater percents of cells that were positive for PD-1 or GADD153. Compared to kidneys of db/m controls, renal cells prepared from kidneys of db/db mice displayed a) increased percent of cells that were positive for IL-17, IL-23, PD-1 and GADD153, b) decreased JC-1 aggregates but increased JC-1 monomers suggestive of disruption of mitochondrial membrane potential and c) increased apoptotic and necrotic cell death. Immunohistochemical analyses also revealed increased staining of renal tissue of db/db mice for IL-17, IL23, GADD153, Annexin V, caspase 3, PD-1 and IDO compared to db/m kidneys; these changes were generally more prominent in the glomeruli. In conclusion, type 2 diabetes upregulates systemic and local ER stress response and pro-inflammatory mechanisms thereby contributing to renal injury. However, the accompanying upregulations of PD-1 and IDO likely reflect activation of compensatory mechanisms to curtail inflammation and cell injury.

Reciprocal relation between GADD153 and Del-1 in regulation of salivary gland inflammation in Sjögren syndrome.

Endoplasmic reticulum (ER) stress response is a pivotal regulator of inflammation and cell death. An integral component of ER stress-induced apoptosis is expression of growth arrest- and DNA damage-inducible protein 153 (GADD153). Further, ER stress response is implicated in leukocyte adhesion and recent studies have discovered endogenous inhibitors of leukocyte adhesion including the developmental endothelial locus-1 (Del-1). Accordingly, we tested the hypothesis that Sjögren's syndrome (SS) is associated with increased salivary gland expression of GADD153 and increased leukocyte infiltration in association with decreased Del-1 thereby contributing to inflammation and cell death. We utilized the non-obese diabetic (NOD) mice, a model of SS-like disease, in association with immunostaining and flow cytometry-based studies. Salivary glands of 14-week-old NOD mice displayed a) increased GADD153 expression, b) marked reduction in Del-1, c) inflammatory cell infiltrates including CD3+ T and CD19+ B lymphocytes as well as M1 and M2 macrophages and d) increased pro-inflammatory interleukin (IL)-17 but reduced anti-inflammatory cytokine, IL-10. These changes were accompanied with disruption of mitochondrial membrane potential and significant increase in apoptosis and necrosis of salivary gland cells of NOD than control mice. Our collective observations suggested that GADD153 directly and/or indirectly through downregulation of Del-1 contributes importantly to salivary gland inflammation and cell death. To establish the relevance of GADD153 and Del-1 for the human condition, lower lip biopsy samples of non-SS subjects and those with a diagnosis of SS were subjected to immunohistochemistry. The results show intense GADD153 immunostaining but marked reduction in Del-1 expression in biopsy samples of SS compared to non-SS subjects. Collectively, the results indicate that GADD153 regulates inflammation and cell death in salivary gland in SS. Further, Del-1 expression likely provides a mechanistic link between increased GADD153 and leukocyte infiltration and accompanying inflammation of salivary gland tissue in this condition.

Aryl hydrocarbon receptor agonist, leflunomide, protects the ischemic-reperfused kidney: role of Tregs and stem cells.

The aryl hydrocarbon receptor (AHR) has emerged as a major modulator of inflammatory processes. We tested the hypothesis that AHR activation protects the ischemic-reperfused kidney in association with the suppression of the inflammatory response. Accordingly, male mice were treated with the nondioxin AHR agonist, leflunomide (40 mg/kg ip); vehicle-treated animals served as controls. Thereafter, the right kidney was subjected to an ischemia (45 min)-reperfusion (4 h) insult, while the left kidney served as a sham control. Renal cells prepared from ischemic-reperfused kidneys of leflunomide-treated mice displayed preservation of mitochondrial membrane potential (Ψ(m)) and decreased apoptosis and necrosis compared with vehicle-treated ischemic-reperfused kidneys. Leflunomide treatment increased regulatory T cells (Tregs; forkhead box P3+) and IL-10-positive cells but reduced IL-17- and IL-23-expressing cells in both the peripheral blood and kidney cells, indicative of down-regulation of inflammatory responses. Leflunomide treatment also increased mobilization of stems cells subsets (i.e., mesenchymal and hematopoietic stem cells and endothelial progenitor cells) in the peripheral blood and promoted their recruitment into the ischemic-reperfused kidney. Collectively, the results indicate that AHR stimulation may represent a novel renoprotective mechanism likely involving mobilization and recruitment of Tregs and stem cells into the damaged kidney.

Renal distal tubule proliferation and increased aquaporin 2 level but decreased urine osmolality in db/db mouse: treatment with chromium picolinate.

Hallmark features of type 2 diabetes mellitus include glucosuria and polyuria. Further, renal aquaporin 2 is pivotal to regulation of fluid excretion and urine osmolality. Accordingly, we tested the hypothesis that the db/db mouse displays increased glucosuria and fluid excretion but reduced urine osmolality in association with decreased renal aquaporin 2 level. In addition, we examined the effect of chromium picolinate (Cr(pic)3) which is purported to improve glycemic control. The db/db mice excreted more urine in association with marked glucose excretion but lower urine osmolality than db/m control group. Light microscopic examination of renal tissue revealed proliferation of tubular structures in db/db compared to the db/m mice, a feature validated with Ki67 immunostaining. Further, these tubules showed generally similar immunostaining intensity and pattern for aquaporin 2 indicating that proliferated tubules are of distal origin. On the other hand, renal aquaporin 2 protein level was significantly higher in the db/db than db/m group. Treatment of db/db mice with Cr(pic)3 reduced plasma glucose and hemoglobin A1c (~15-17%, p<0.05) and Ki67 positive cells but other parameters were similar to their untreated counterparts. Collectively, these findings suggest that proliferation of renal distal tubules and increased aquaporin 2 level likely represent an adaptive mechanism to regulate fluid excretion to prevent dehydration in the setting of marked glucosuria in the db/db mouse, features not affected by Cr(pic)3 treatment. These observations are of relevance to increasing interest in developing therapeutic agents that facilitate renal glucose elimination.

Use of a new, simple, laboratory method for screening the antimicrobial and antiviral properties of hand sanitizers.

PURPOSE: To develop a simple, laboratory method for screening the antimicrobial/antiviral activity of hand sanitizers, to replace the more time consuming use of human volunteers. METHODS: A Rapid Agar Plate Assay (RAPA) was developed that uses sterile agar plates to simulate skin surfaces. After treating the agar plates with putative hand sanitizers, the plates were inoculated with gram-positive S. aureus or gram-negative E. coli. Untreated agar plates served as controls. After incubation for 48 hours, the bacteria were recovered and stained with fluorescent dyes. The number of live/dead bacteria was quantitated by flow cytometry. For anti-viral activity, mammalian cell lines were grown to confluency and infected with noroviruses (murine norovirus or feline calicivirus), and the number of dead cells was quantitated as the log10 of number of cells killed. A liquid hand soap without any antibacterial activity (LHS) was used as the control. A popular ethanol-based hand sanitizer (GHS) was compared to a new quaternary ammonium-containing bactericidal hand cream (ABC). RESULTS: The liquid soap was not effective against either gram-positive or gram-negative bacteria, or viruses. Both GHS and ABC were very effective against S. aureus, but much less so against E. coli. Both GHS and ABC were even more effective against the two noroviruses that cause gastrointestinal diseases, than they were against gram-positive bacteria. These results support the use of RAPA as an effective laboratory screening test to evaluate the antibacterial/antiviral activity of hand sanitizers or other antimicrobial products.

Mitochondrial complex I and NAD(P)H oxidase are major sources of exacerbated oxidative stress in pressure-overloaded ischemic-reperfused hearts.

We tested the hypothesis that pressure overload exacerbates oxidative stress associated with augmented mitochondrial permeability transition (MPT) pore opening and cell death in ischemic-reperfused hearts. Pressure overload decreased the level of reduced glutathione but increased nitrotyrosine and 8-hydroxydeoxyguanosine levels in ischemic-reperfused hearts. The activity of catalase, but not superoxide dismutase (SOD), was lower in ischemic-reperfused hearts perfused at higher pressure. Mitochondria from ischemic-reperfused hearts subjected to higher perfusion pressure displayed significantly greater [³H]-2-deoxyglucose-6-P entrapment suggestive of greater MPT pore opening and consistent with greater necrosis and apoptosis. Tempol (SOD mimetic) reduced infarct size in both groups but it remained greater in the higher pressure group. By contrast, uric acid (peroxynitrite scavenger) markedly reduced infarct size at higher pressure, effectively eliminating the differential between the two groups. Inhibition of xanthine oxidase, with allopurinol, reduced infarct size but did not eliminate the differential between the two groups. However, amobarbital (inhibitor of mitochondrial complex I) or apocynin [inhibitor of NAD(P)H oxidase] reduced infarct size at both pressures and also abrogated the differential between the two groups. Consistent with the effect of apocynin, pressure-overloaded hearts displayed significantly higher NAD(P)H oxidase activity. Furthermore, pressure-overloaded hearts displayed increased nitric oxide synthase activity which, along with increased propensity to superoxide generation, may underlie uric acid-induced cardioprotection. In conclusion, increased oxidative and nitrosative stress, coupled with lack of augmented SOD and catalase activities, contributes importantly to the exacerbating impact of pressure overload on MPT pore opening and cell death in ischemic-reperfused hearts.

Submandibular gland and caries susceptibility in the obese Zucker rat.

BACKGROUND: Obesity is a prevalent disorder characterized as marked insulin resistance and low grade inflammation. We tested the hypothesis that obesity upregulates inflammatory markers in the submandibular gland in association with derangements of its architecture and pre-disposition to caries in obese Zucker rats (OZR). We also examined the potential impact of chromium picolinate (Cr(Pic)3), a nutritional supplement suggested to improve glycemic control, on the aforementioned parameters. DESIGN: Male OZR were treated with diets lacking and containing 5 or 10 mg/kg chromium (as Cr(Pic)3) from 6 weeks to about 6 months of age; lean Zucker rats (LZR) served as controls. Thereafter, glycemic status, salivary tissue architecture, and the levels of several inflammatory markers were determined in association with caries susceptibility. RESULTS: OZR showed reduced insulin sensitivity, increased ratio of phospho-nuclear factor-kappa B (NF-κB) to total NF-κB, and increased intercellular adhesion molecule-1 level but similar histological features compared to LZR. Importantly, compared to LZR, OZR displayed rampant caries and a tendency for reduced dentin mineral density. Treatment of OZR with Cr(Pic)3 attenuated upregulation of these proinflammatory indicators in association with reduced severity of caries without improving insulin sensitivity. CONCLUSIONS: Obesity promotes proinflammatory changes within the submandibular gland, without affecting glandular architecture, in association with rampant caries; Cr(Pic)3 treatment provided some protective effects.

Differential effects of taurine treatment and taurine deficiency on the outcome of renal ischemia reperfusion injury.

Taurine possesses membrane stabilization, osmoregulatory and antioxidant properties, aspects of relevance to ischemic injury. We tested the hypothesis that body taurine status is a determinant of renal ischemic injury. Accordingly, renal function and structure were examined in control (C), taurine-treated (TT) and taurine deficient (TD) rats that were subjected to bilateral renal ischemia (60 min) followed by reperfusion (IR); sham operated rats served as controls. Baseline urine osmolality was greater in the TD group than in the control and the TT groups, an effect associated with increased renal aquaporin 2 level. The IR insult reduced urine osmolality (i.e., day-1 post insult); the TD/IR group displayed a more marked recovery in urine osmolality by day-6 post insult than the other two groups. Fluid and sodium excretions were lower in the TD/IR group, suggesting propensity to retention. Histopathological examination revealed the presence of tubular necrotic foci in the C/IR group than sham controls. While renal architecture of the TD/IR group showed features resembling sham controls, the TT/IR group showed dilated tubules, which lacked immunostaining for aquaporin 2, but not 1, suggestive of proximal tubule origin. Finally, assessment of cell proliferation and apoptosis revealed lower proliferation but higher apoptotic foci in the TT/IR group than other IR groups. Collectively, the results indicate that body taurine status is a major determinant of renal IR injury.

Effects of artificial light at night on foraging behavior and vigilance in a nocturnal rodent.

Artificial light at night has greatly changed the physical environment for many organisms on a global scale. As an energy efficient light resource, light emitting diodes (LEDs) have been widely used in recent years. As LEDs often have a broad spectrum, many biological processes may be potentially affected. In this study, we conducted manipulated experiments in rat-proof enclosures to explore the effects of LED night lighting on behavior of a nocturnal rodent, the Mongolian five-toed jerboa (Allactaga sibirica). We adopted the giving-up density (GUD) method and camera video trapping to study behavioral responses in terms of patch use, searching efficiency and vigilance. With the presence of white LED lighting, jerboas spent less time in patches, foraged less intensively (with higher GUDs) and became vigilant more frequently, while their searching efficiency was higher than under dark treatment. Although both positive and negative effects of LEDs on foraging were detected, the net effect of LEDs on jerboas is negative, which may further translate into changes in population dynamics, inter-specific interaction and community structure. To our knowledge, this is the first field study to explore how LED lighting affect foraging behavior and searching efficiency in rodents. Our results may have potential implications for practices such as pest control.

Status of stem cells in diabetic nephropathy: predictive and preventive potentials.

BACKGROUND: Recruitment of stem cells to sites of tissue injury constitutes an important mechanism aimed at tissue repair and regeneration. However, it is not clear how the diabetic milieu affects the viability of endogenous stem cells. Thus, we tested the hypothesis that diabetes mellitus is associated with increased apoptosis which, in turn, contributes to reduction in stem cells and the manifestation of type 2 diabetic nephropathy. METHODS: Sixteen-week-old male obese type 2 diabetic db/db mice, and their appropriate controls, were used for assessment of the status of endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and hematopoetic stem cells (HSCs) in the peripheral blood and renal tissue using specific cell markers. Further, we explored whether diabetic animals display greater apoptosis of stem cell subsets. RESULTS: The peripheral blood cells of db/db mice displayed reduction in EPCs (p < 0.05) compared to those of db/m controls. Further, kidney cells prepared from experimental groups also showed reductions in EPCs, MSCs, and HSCs. We also observed increased apoptosis of stem cell subsets in cells prepared from kidneys of db/db than those of db/m mice. CONCLUSIONS: The present study shows a similar pattern of decline in stem cell subsets in peripheral blood and kidneys of db/db mice, an effect likely related to increased apoptosis. Collectively, the results suggest that apoptosis of stem cells likely contributes to eventual manifestation of renal failure in diabetes mellitus. Monitoring of blood levels of stem cell subsets could predict failure of their reparative and protective effects and eventual manifestations of diabetic complications.

Upregulation of Programmed Death-1 and Its Ligand in Cardiac Injury Models: Interaction with GADD153.

PURPOSE: Programmed Death-1 (PD-1) and its ligand, PD-L1, are regulators of immune/ inflammatory mechanisms. We explored the potential involvement of PD-1/PD-L1 pathway in the inflammatory response and tissue damage in cardiac injury models. EXPERIMENTAL DESIGN: Ischemic-reperfused and cryoinjured hearts were processed for flow cytometry and immunohistochemical studies for determination of cardiac PD-1 and PD-L1 in the context of assessment of the growth arrest- and DNA damage-inducible protein 153 (GADD153) which regulates both inflammation and cell death. Further, we explored the potential ability of injured cardiac cells to influence proliferation of T lymphocytes. RESULTS: The isolated ischemic-reperfused hearts displayed marked increases in expression of PD-1 and PD-L1 in cardiomyocytes; however, immunofluorescent studies indicate that PD-1 and PD-L1 are not primarily co-expressed on the same cardiomyocytes. Upregulation of PD-1/PD-L1 was associated with a) marked increases in GADD153 and interleukin (IL)-17 but a mild increase in IL-10 and b) disruption of mitochondrial membrane potential (ψm) as well as apoptotic and necrotic cell death. Importantly, while isotype matching treatment did not affect the aforementioned changes, treatment with the PD-L1 blocking antibody reversed those effects in association with marked cardioprotection. Further, ischemic-reperfused cardiac cells reduced proliferation of T lymphocytes, an effect partially reversed by PD-L1 antibody. Subsequent studies using the cryoinjury model of myocardial infarction revealed significant increases in PD-1, PD-L1, GADD153 and IL-17 positive cells in association with significant apoptosis/necrosis. CONCLUSIONS: The data suggest that upregulation of PD-1/PD-L1 pathway in cardiac injury models mediates tissue damage likely through a paracrine mechanism. Importantly, inhibition of T cell proliferation by ischemic-reperfused cardiac cells is consistent with the negative immunoregulatory role of PD-1/PD-L1 pathway, likely reflecting an endogenous cardiac mechanism to curtail the deleterious impact of infiltrating immune cells to the damaged myocardium. The balance of these countervailing effects determines the extent of cardiac injury.

The status of glucocorticoid-induced leucine zipper protein in the salivary glands in Sjögren's syndrome: predictive and prognostic potentials.

BACKGROUND: We recently showed that an imbalance between the pro-inflammatory cytokine, interleukin (IL)-17, and the developmental endothelial locus-1 (Del-1) likely contributes to inflammation and salivary gland abnormalities in Sjögren's syndrome (SS). The glucocorticoid-induced leucine zipper (GILZ) protein is a pivotal player in mediating the anti-inflammatory effects of glucocorticoids. However, its status and role in salivary gland inflammation and dysfunction in SS are not established. Thus, we tested the hypothesis that SS is associated with reduced GILZ expression, thereby contributing to Del-1/Il-17 imbalance and inflammation in salivary glands. METHODS: We utilized the nonobese diabetic (NOD) mice, a model of SS-like disease as well as lower-lip biopsy samples of subjects without or with a diagnosis of SS in association with immunostaining studies. These studies were complemented with in vitro and flow-cytometry studies whereby interleukin (IL)-23-treated salivary gland cells were co-cultured with GILZ-expressing cells or control cells; IL-23 is known to increase generation of IL-17. RESULTS: Salivary glands of NOD mice displayed marked leukocyte infiltration and reduced GILZ expression in association with increased IL-17 but decreased Del-1 expression. A similar pattern was observed for lower-lip biopsy samples of SS than non-SS subjects. Further, IL-23-treated salivary gland cells displayed marked increase in IL-17 but reduced Del-1 positive cells which were reversed with co-culturing with GILZ-expressing cells but not control cells. Collectively, the results are suggestive of dysregulation of GILZ playing a role in inflammation and associated Del-1/Il-17 imbalance in SS. CONCLUSIONS: Complementing our demonstration of Del-1/IL-17 imbalance in salivary glands in SS, the present study has established the relevance and significance of GILZ as a novel predictive and prognostic molecular fingerprint for SS. Thus, assessment of minor salivary gland GILZ expression, in conjunction with Del-1/IL-17 imbalance, could potentially offer a more sensitive approach to help with diagnosis of SS, at early stage of the disease, than that based on leukocyte infiltration. Future studies should establish whether treatment with GILZ ameliorates signs and symptoms of salivary malfunction of SS for which effective treatment options remain elusive.

SGK-1 regulates inflammation and cell death in the ischemic-reperfused heart: pressure-related effects.

BACKGROUND: Systemic hypertension and the associated increased myocardial load/mechanical stress are common in patients with coronary heart disease. Thus, unraveling of mechanosensitive molecular mechanisms that determine cell fate in the setting of cardiac tissue injury is of scientific and clinical relevance. We tested the hypothesis that the prosurvival, mechanosensitive, serum glucocorticoid-regulated kinase-1 (SGK-1) is a pivotal determinant of pressure-related inflammatory response and cell fate in the ischemic-reperfused heart. METHODS: Langendorff-perfused rat hearts were subjected to an ischemia reperfusion (IR) insult, at 80 or 160cm water, with perfusate lacking or containing the SGK-1 inhibitor GSK650394A (1 µM); normoxic hearts served as controls. Thereafter, hearts tissues were used for Western blotting or cardiac cells were prepared for flow cytometry and immunofluorescent studies. RESULTS: An IR insult (i) reduced phosphoSGK-1 (active and protective) in association with disruption of mitochondrial membrane potential (ψm) and increased apoptosis and necrosis and (ii) increased expressions of growth-arrest and DNA damage-associated protein 153 (GADD153; a determinant of inflammation and cell death) and the proinflammatory cytokine interleukin (IL) 17; these effects were greater at high pressure. On the other hand, the anti-inflammatory cytokines IL-10 and IL-27 increased more in ischemic-reperfused hearts subjected to low pressure. SGK-1 inhibition further reduced phosphoSGK-1, increased GADD153 and IL-17, and reduced IL-10 and IL-27 in association with augmented disruption of ψm and exacerbated cell death; these effects were greater at low pressure. CONCLUSIONS: The results indicate a major pressure-related role for SGK-1 in regulating inflammation and cell fate in the ischemic-reperfused heart.

The role of indoleamine 2,3 dioxygenase in beneficial effects of stem cells in hind limb ischemia reperfusion injury.

Ischemia-Reperfusion (IR) injury of limb remains a significant clinical problem causing secondary complications and restricting clinical recovery, despite rapid restoration of blood flow and successful surgery. In an attempt to further improve post ischemic tissue repair, we investigated the effect of a local administration of bone marrow derived stem cells (BMDSCs) in the presence or absence of immune-regulatory enzyme, IDO, in a murine model. A whole limb warm ischemia-reperfusion model was developed using IDO sufficient (WT) and deficient (KO) mice with C57/BL6 background. Twenty-four hours after injury, 5 × 105 cells (5×105 cells/200 µL of PBS solution) BMDSCs (Sca1 + cells) were injected intramuscularly while the control group received just the vehicle buffer (PBS). Forty-eight to seventy-two hours after limb BMDSC injection, recovery status including the ratio of intrinsic paw function between affected and normal paws, general mobility, and inflammatory responses were measured using video micrometery, flow cytometry, and immunohistochemistry techniques. Additionally, MRI/MRA studies were performed to further study the inflammatory response between groups and to confirm reconstitution of blood flow after ischemia. For the first time, our data, showed that IDO may potentially represent a partial role in triggering the beneficial effects of BMDSCs in faster recovery and protection against structural changes and cellular damage in a hind limb IR injury setting (P = 0.00058).

Targeting serum glucocorticoid-regulated kinase-1 in squamous cell carcinoma of the head and neck: a novel modality of local control.

PURPOSE: The inhibition of serum glucocorticoid-regulated kinase-1 (SGK-1) has been found to decrease growth of colon and prostate cancer cells. The purpose of this study is to evaluate the therapeutic effect of SGK-1 inhibition in head and neck squamous cell carcinoma (SCC). EXPERIMENTAL DESIGN: Human head and neck tumors (HTB41/43) were established in athymic mice. Growth rates between mice treated with vehicle (PBS) injection (group 1, n = 5), SGK-1 Inhibitor GSK 650394 (group 2, n = 6), systemic cisplatin (group 3, n = 6), and a combination of SGK-1 Inhibitor and cisplatin (group 4, n = 6) were compared using repeated measures one-way ANOVA with Newman-Keuls Multiple Comparison Test. Tumor cells were subsequently submitted to further analyses. RESULTS: At the end of the experiment mean tumor sizes were 122.33+/-105.86, 76.73+/-36.09, 94.52+/-75.92, and 25.76+/-14.89 mm2 (mean +/- SD) for groups 1 to 4. Groups 2 and 3 showed decreased tumor growth compared to controls (p<0.001). Group 4 displayed even greater growth suppression (p<0.0001). Importantly, group 4 fared better than group 3 (p<0.001). CD44 expression was reduced in group 2 (p<0.05), and to an even greater extent in groups 3 and 4 (p<0.0025). A trend towards reduction of HER 2 expression was noted in group 4. CONCLUSIONS: SGK-1 inhibition suppresses tumor growth, and in combination with systemic cisplatin exceeds the effect of cisplatin alone. Decreased expression of CD44 and HER 2 implies depletion of tumor stem cells, and less tumorigenicity. SGK-1 inhibition represents a potential modality of local control for palliation in advanced cases.

Pressure overload regulates expression of cytokines, γH2AX, and growth arrest- and DNA-damage inducible protein 153 via glycogen synthase kinase-3ß in ischemic-reperfused hearts.

The growth arrest- and DNA-damage inducible protein 153 (GADD153) regulates both apoptosis and inflammatory response. Importantly, glycogen synthase kinase-3ß (GSK-3ß) may provide a mechanistic link for cellular expression of GADD153, inflammatory response, and cell death. We previously showed that pressure overload exacerbates myocardial ischemia reperfusion injury associated with significant reduction in phosphorylated (inactive) GSK-3ß. This raises the possibility that pressure overload, through a GSK-3ß-dependent mechanism, increases GADD153 expression, thereby upregulating inflammatory cytokine production and contributing to worsening of myocardial ischemia reperfusion injury. Accordingly, Langendorff-perfused rat hearts were subjected to global ischemia reperfusion protocol in the absence or presence of the GSK-3ß inhibitor, lithium chloride (1 mmol/L), with perfusion pressure set at 80 or 160 cmH(2)O; normoxic hearts served as controls. Compared with normoxia, an ischemia reperfusion insult increased expressions of proinflammatory cytokines, γH2AX, and GADD153 in association with increased cell death. In the ischemic-reperfused hearts, pressure overload did the following: (1) reduced interleukin-10 but increased interleukin-17 (cardiomyocytes), without affecting interleukin-23; (2) increased expressions of γH2AX and GADD153; (3) decreased 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) aggregates but increased JC-1 monomers (suggestive of reduced mitochondrial membrane potential, ψ(m)); and (4) increased annexin V immunostaining as well as apoptotic and necrotic cell death. Treatment with lithium chloride caused a robust increase in interleukin-10, preserved ψ(m), and markedly decreased all other parameters with the effect being most prominent for hearts perfused at the high pressure. In conclusion, pressure overload, via a GSK-3ß-dependent mechanism, exacerbates cell death in the isolated ischemic-reperfused heart involving regulation of inflammatory response, DNA injury, and GADD153 expression.

Mechanisms of load dependency of myocardial ischemia reperfusion injury.

Coronary artery disease and associated ischemic heart disease are prevalent disorders worldwide. Further, systemic hypertension is common and markedly increases the risk for heart disease. A common denominator of systemic hypertension of various etiologies is increased myocardial load/mechanical stress. Thus, it is likely that high pressure/mechanical stress attenuates the contribution of cardioprotective but accentuates the contribution of cardiotoxic pathways thereby exacerbating the outcome of an ischemia reperfusion insult to the heart. Critical events which contribute to cardiomyocyte injury in the ischemic-reperfused heart include cellular calcium overload and generation of reactive oxygen/nitrogen species which, in turn, promote the opening of the mitochondrial permeability transition pore, an important event in cell death. Increasing evidence also indicates that the myocardium is capable of mounting a robust inflammatory response which contributes importantly to tissue injury. On the other hand, cardioprotective maneuvers of ischemic preconditioning and postconditioning have led to identification of complex web of signaling pathways (e.g., reperfusion injury salvage kinase) which ultimately converge on the mitochondria to exert cytoprotection. The present review is intended to briefly describe mechanisms of cardiac ischemia reperfusion injury followed by a discussion of our work focused on how pressure/mechanical stress modulates endogenous cardiotoxic and cardioprotective mechanisms to ultimately exacerbate ischemia reperfusion injury.

Renal and glycemic effects of high-dose chromium picolinate in db/db mice: assessment of DNA damage.

This study examined renal and glycemic effects of chromium picolinate [Cr(pic)3] supplementation in the context of its purported potential for DNA damage. In preventional protocol, male obese diabetic db/db mice were fed diets either lacking or containing 5, 10 or 100 mg/kg chromium as Cr(pic)3 from 6 to 24 weeks of age; male lean nondiabetic db/m mice served as controls. Untreated db/db mice displayed increased plasma glucose and insulin, hemoglobin A1c, renal tissue advanced glycation end products, albuminuria, glomerular mesangial expansion, urinary 8-hydroxydeoxyguanosine (an index of oxidative DNA damage) and renal tissue immunostaining for γH2AX (a marker of double-strand DNA breaks) compared to db/m controls. Creatinine clearance was lower in untreated db/db mice than their db/m controls, while blood pressure was similar. High Cr(pic)3 intake (i.e., 100-mg/kg diet) mildly improved glycemic status and albuminuria without affecting blood pressure or creatinine clearance. Treatment with Cr(pic)3 did not increase DNA damage despite marked renal accumulation of chromium. In interventional protocol, effects of diets containing 0, 100 and 250 mg/kg supplemental chromium, from 12 to 24 weeks of age, were examined in db/db mice. The results generally revealed similar effects to those of the 100-mg/kg diet of the preventional protocol. In conclusion, the severely hyperglycemic db/db mouse displays renal structural and functional abnormalities in association with DNA damage. High-dose Cr(pic)3 treatment mildly improves glycemic control, and it causes moderate reduction in albuminuria, without affecting the histopathological appearance of the kidney and increasing the risk for DNA damage.

Effect of pressure overload on cardioprotection via PI3K-Akt: comparison of postconditioning, insulin, and pressure unloading.

BACKGROUND: Postconditioning (PC) and insulin exert cardioprotection by activating phosphatidylinositol-3 kinase (PI3K) signaling. Because pressure overload exacerbates ischemia-reperfusion (IR) injury, we tested the hypothesis that (i) pressure overload attenuates PC- and insulin-induced cardioprotection, an effect caused by reduced PI3K-Akt signaling and (ii) pressure unloading confers cardioprotection comparable to either PC or insulin. METHODS: Infarct size (IS) and levels of relevant proteins (i.e., Akt, glycogen synthase kinase-3beta (GSK-3beta), 3'-phosphoinositide dependent kinase 1 (PDK1), phosphatase and tensin homolog on chromosome ten (PTEN)) were determined in hearts subjected to IR. RESULTS: Pressure overload increased IS in association with changes in protein levels consistent with reduced PI3K-Akt signaling (i.e., ischemic reperfused vs. normoxic hearts). PC and insulin reduced IS but it was greater in hearts perfused at the higher, than the lower, pressure. Wortmannin (PI3K inhibitor) partially reversed PC-induced cardioprotection, with IS being greater in the high-pressure group. Pressure unloading during reperfusion caused the most marked reduction in IS whereas pressure loading abolished PC-induced cardioprotection. Nonetheless, the phospho-Akt/total Akt ratios and phospho-GSK-3beta levels were unaffected by perfusion pressure in insulin-treated or postconditioned hearts. Moreover, protein levels were similar in pressure-unloaded and pressure-loaded hearts. CONCLUSIONS: Pressure overload reduces PI3K-Akt signaling following IR. However, a differential in PI3K-Akt signaling was not observed in ischemia-reperfused, insulin-treated, and postconditioned hearts, suggesting involvement of pathways other than PI3K-Akt for the effect of pressure on IS. Importantly, pressure unloading at reperfusion represents a novel and effective cardioprotective maneuver.