Therapy of murine squamous cell carcinomas with 2-difluoromethylornithine

Targeted overexpression of an ornithine decarboxylase (ODC) transgene to mouse skin (the K6/ODC mouse) significantly enhances susceptibility to carcinogenesis. While in most strain backgrounds the predominant tumor type resulting from initiation-promotion protocols is benign squamous papilloma, K6/ODC mice on a FVB/N background develop malignant squamous cell carcinomas (SCCs) rapidly and in high multiplicity after carcinogen treatment. We have investigated the utility of polyamine-based therapy against SCCs in this model using the ODC inhibitor 2-difluoromethylornithine delivered orally. At a 2% concentration in drinking water, DFMO caused rapid tumor regression, but in most cases, tumors eventually regrew rapidly even in the presence of DFMO. The tumors that regrew were spindle cell carcinomas, an aggressive undifferentiated variant of SCC. At 1% DFMO in the drinking water, tumors also responded rapidly, but tumor regrowth did not occur. The majority of DFMO-treated SCCs were classified as complete responses, and in some cases, apparent tumor cures were achieved. The enzymatic activity of ODC, the target of DFMO, was substantially reduced after treatment with 1% DFMO and the high SCC polyamine levels, especially putrescine, were also significantly lowered. Based on the results of BrdUrd labeling and TUNEL assays, the effect of DFMO on SCC growth was accompanied by a significant reduction in tumor proliferation with no increase in the apoptotic index. These results demonstrate that SCCs, at least in the mouse, are particularly sensitive to polyamine-based therapy.     

Inhibition of p38 MAPK Signaling Augments Skin Tumorigenesis via NOX2 Driven ROS Generation

p38 mitogen-activated protein kinases (MAPKs) respond to a wide range of extracellular stimuli. While the inhibition of p38 signaling is implicated in the impaired capacity to repair ultraviolet (UV)-induced DNA damage—a primary risk factor for human skin cancers—its mechanism of action in skin carcinogenesis remains unclear, as both anti-proliferative and survival functions have been previously described. In this study, we utilized cultured keratinocytes, murine tumorigenesis models, and human cutaneous squamous cell carcinoma (SCC) specimens to assess the effect of p38 in this regard. UV irradiation of normal human keratinocytes increased the expression of all four p38 isoforms (α/β/γ/δ); whereas irradiation of p53-deficient A431 keratinocytes derived from a human SCC selectively decreased p38α, without affecting other isoforms. p38α levels are decreased in the majority of human cutaneous SCCs assessed by tissue microarray, suggesting a tumor-suppressive effect of p38α in SCC pathogenesis. Genetic and pharmacological inhibition of p38α and in A431 cells increased cell proliferation, which was in turn associated with increases in NAPDH oxidase (NOX2) activity as well as intracellular reactive oxygen species (ROS). These changes led to enhanced invasiveness of A431 cells as assessed by the matrigel invasion assay. Chronic treatment of p53^-/-/SKH-1 mice with the p38 inhibitor SB203580 accelerated UV-induced SCC carcinogenesis and increased the expression of NOX2. NOX2 knockdown suppressed the augmented growth of A431 xenografts treated with SB203580. These findings indicate that in the absence of p53, p38α deficiency drives SCC growth and progression that is associated with enhanced NOX2 expression and ROS formation.     

Disruption of Protein Kinase A in Mice Enhances Healthy Aging

Mutations that cause a reduction in protein kinase A (PKA) activity have been shown to extend lifespan in yeast. Loss of function of mammalian RIIβ, a regulatory subunit of PKA expressed in brain and adipose tissue, results in mice that are lean and insulin sensitive. It was therefore hypothesized that RIIB null (RIIβ^−/−) mice would express anti-aging phenotypes. We conducted lifespan studies using 40 mutant and 40 wild type (WT) littermates of equal gender numbers and found that both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the 80% lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). There was no difference in either median or 80% lifespan in female genotypes. WT mice of both genders became increasingly obese with age, while mutant mice maintained their lean phenotype into old age. Adiposity was found to correlate with lifespan for males only. 50% of male mice between 30 and 35 g, corresponding to about 5% body fat, for either genotype lived over 1000 days. No male mouse outside of this weight range achieved this lifespan. During their last month of life, WT mice began losing weight (a total of 8% and 15% of body weight was lost for males and females, respectively), but RIIβ^−/− male mice maintained their lean body mass to end of life. This attenuation of decline was not seen in female mutant mice. Old male mutant mice were insulin sensitive throughout their life. Both genders showed modestly lower blood glucose levels in old mutants compared to WT. Male mutants were also resistant to age-induced fatty liver. Pathological assessment of tissues from end of life male mutant mice showed a decrease in tumor incidence, decreased severity of renal lesions, and a trend towards a decrease in age-related cardiac pathology. These findings help establish the highly conserved nature of PKA and suggest that disruption of PKA affects physiological mechanisms known to be associated with healthy aging.     

The oncogenic role of GASC1 in chemically induced mouse skin cancer

Gene amplified in squamous cell carcinoma (SCC) 1 (GASC1), also known as KDM4C/JMJD2C, encodes a histone demethylase that specifically demethylates lysine residues (H3K9, H3K36, and H1.4K26) and plays a crucial role in the regulation of gene expression as well as in heterochromatin formation. GASC1 is located at human chromosome 9p23–24, where frequent genomic amplification is observed in human esophageal cancer, and its aberrant expression is detected in a variety of human cancers, such as breast, colon, and prostate. Therefore, it is highly likely that GASC1 contributes to the genesis and/or development of cancer. However, there is a lack of direct evidence of GASC1 having an oncogenic function. In this study, we aimed to clarify the role of GASC1 in the skin SCC carcinogenesis. For this purpose, we generated Gasc1-heterozygous mice (Gasc1 ^+/−) with reduced expression of Gasc1. On the basis of our results, Gasc1 ^+/− mice displayed a significantly lower incidence and multiplicity of both benign and malignant tumors induced by the two-stage skin carcinogenesis protocol than wild-type mice. In addition, the volume of carcinoma was significantly lower in Gasc1 ^+/− mice. Consistent with these observations, knocking down of Gasc1 resulted in reduced cell viability of SCC cells in vitro. Our findings clearly demonstrated that GASC1 has an oncogenic role in skin carcinogenesis.

     

RSK Activation of Translation Factor eIF4B Drives Abnormal Increases of Laminin γ2 and MYC Protein during Neoplastic Progression to Squamous Cell Carcinoma

Overexpression of the basement membrane protein Laminin γ2 (Lamγ2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs). This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown. We recently reported that Lamγ2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade. Here we identify eIF4B as the RSK downstream effector responsible for elevated Lamγ2 as well as MYC protein in neoplastic epithelial cells. Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the E6 oncoprotein of human papillomavirus (HPV) type 16 displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lamγ2 and MYC in culture. Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression. However, pharmacologic kinase inhibitor studies in culture revealed that Lamγ2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1. eIF4B knockdown reduced Lamγ2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5′ untranslated regions (5′-UTRs). Accordingly, expression of a luciferase reporter construct preceded by the Lamγ2 5′-UTR proved to be RSK-dependent and mTOR-independent. These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lamγ2 and MYC protein levels during neoplastic progression to invasive SCC. These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC.     

ABO Blood Group and Incidence of Skin Cancer

Background

Previous studies have examined the association between ABO blood group and the risk of some malignancies. However, no prospective cohort study to date has examined the association between ABO blood group and the risk of skin cancer.

Methodology/Principal Findings

Using two large cohorts in the US, we examined ABO blood type and incidence of skin cancer, including melanoma, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC). We followed up study participants (70,650 female nurses and 24,820 male health professionals) on their diagnosis of incident skin cancer from cohort baseline (1976 in women and 1986 in men) until 2006. Study participants reported their blood type in 1996 in both cohorts. During the follow-up, 685 participants developed melanoma, 1,533 developed SCC and 19,860 developed BCC. We used Cox proportional hazards models to calculate the hazard ratios (HR) and 95% confidence intervals (CI) of each type of skin cancer. We observed that non-O blood group (A, AB, and B combined) was significantly associated with a decreased risk of non-melanoma skin cancer overall. Compared to participants with blood group O, participants with non-O blood group had a 14% decreased risk of developing SCC (multivariable HR: 0.86; 95% CI: 0.78, 0.95) and a 4% decreased risk of developing BCC (multivariable HR: 0.96; 95% CI: 0.93, 0.99). The decreased risk of melanoma for non-O blood group was not statistically significant (multivariable HR: 0.91; 95% CI: 0.78, 1.05).

Conclusion/Significance

In two large independent populations, non-O blood group was associated with a decreased risk of skin cancer. The association was statistically significant for non-melanoma skin cancer. Additional studies are needed to confirm these associations and to define the mechanisms by which ABO blood type or closely linked genetic variants may influence skin cancer risk.     

Impact of regulatory light chain mutation K104E on the ATPase and motor properties of cardiac myosin

Mutations in the cardiac myosin regulatory light chain (RLC, MYL2 gene) are known to cause inherited cardiomyopathies with variable phenotypes. In this study, we investigated the impact of a mutation in the RLC (K104E) that is associated with hypertrophic cardiomyopathy (HCM). Previously in a mouse model of K104E, older animals were found to develop cardiac hypertrophy, fibrosis, and diastolic dysfunction, suggesting a slow development of HCM. However, variable penetrance of the mutation in human populations suggests that the impact of K104E may be subtle. Therefore, we generated human cardiac myosin subfragment-1 (M2β-S1) and exchanged on either the wild type (WT) or K104E human ventricular RLC in order to assess the impact of the mutation on the mechanochemical properties of cardiac myosin. The maximum actin-activated ATPase activity and actin sliding velocities in the in vitro motility assay were similar in M2β-S1 WT and K104E, as were the detachment kinetic parameters, including the rate of ATP-induced dissociation and the ADP release rate constant. We also examined the mechanical performance of α-cardiac myosin extracted from transgenic (Tg) mice expressing human wild type RLC (Tg WT) or mutant RLC (Tg K104E). We found that α-cardiac myosin from Tg K104E animals demonstrated enhanced actin sliding velocities in the motility assay compared with its Tg WT counterpart. Furthermore, the degree of incorporation of the mutant RLC into α-cardiac myosin in the transgenic animals was significantly reduced compared with wild type. Therefore, we conclude that the impact of the K104E mutation depends on either the length or the isoform of the myosin heavy chain backbone and that the mutation may disrupt RLC interactions with the myosin lever arm domain.     

RanBP9 aggravates synaptic damage in the mouse brain and is inversely correlated to spinophilin levels in Alzheimer's brain synaptosomes

We previously demonstrated that overexpression of RanBP9 led to enhanced Aβ generation in a variety of cell lines and primary neuronal cultures, and subsequently, we confirmed increased amyloid plaque burden in a mouse model of Alzheimer''s disease (AD). In the present study, we found striking reduction of spinophilin protein levels when RanBP9 is overexpressed. At 12 months of age, we found spinophilin levels reduced by 70% (P<0.001) in the cortex of APΔE9/RanBP9 mice compared with that in wild-type (WT) controls. In the hippocampus, the spinophilin levels were reduced by 45% (P<0.01) in the APΔE9/RanBP9 mice. Spinophilin immunoreactivity was also reduced by 22% (P<0.01) and 12% (P<0.05) in the cortex of APΔE9/RanBP9 and APΔE9 mice, respectively. In the hippocampus, the reductions were 27% (P<0.001) and 14% (P<0.001) in the APΔE9/RanBP9 and APΔE9 mice, respectively. However, in the cerebellum, spinophilin levels were not altered in either APΔE9 or APΔE9/RanBP9 mice. Additionally, synaptosomal functional integrity was reduced under basal conditions by 39% (P<0.001) in the APΔE9/RanBP9 mice and ∼23% (P<0.001) in the APΔE9 mice compared with that in WT controls. Under ATP- and KCl-stimulated conditions, we observed higher mitochondrial activity in the WT and APΔE9 mice, but lower in the APΔE9/RanBP9 mice. Significantly, we confirmed the inverse relationship between RanBP9-N60 and spinophilin in the synaptosomes of Alzheimer''s brains. More importantly, both APΔE9 and APΔE9/RanBP9 mice showed impaired learning and memory skills compared to WT controls. These data suggest that RanBP9 might play a crucial role in the loss of spines and synapses in AD.     

GSDME deficiency leads to the aggravation of UVB-induced skin inflammation through enhancing recruitment and activation of neutrophils

Gasdermin E (GSDME)-mediated pyroptosis is induced in keratinocytes of UVB-challenged skin. The role of GSDME in UVB-caused skin damage remains unknown. To explore the role of GSDME in UVB-induced skin inflammation. We compared differences in skin appearance, histological features, keratinocyte death modalities, infiltration of immune cells, and levels of some inflammatory cytokines between Gsdme^−/− mice and wild type (WT) mice after UVB exposure. We explored whether keratinocytes contribute to GSDME deficiency-caused aggravation of UVB-induced skin inflammation in GSDME knockdown keratinocyte cultured in vitro and keratinocyte-specific Gsdme conditional knockout mice. We used anti-Ly6G antibody to deplete neutrophils and explore their role in UVB-caused skin damage. Skin damage and neutrophils infiltration were aggravated in UVB-challenged Gsdme^−/− mice, compared with UVB-challenged WT mice. Apoptosis and necroptosis, which were initiated together with GSDME-mediated pyroptosis in UVB-challenged WT mice, were not enhanced in UVB-challenged Gsdme^−/− mice. Neutrophils activation indicators and its recruiting cytokines were increased in skin tissue of UVB-challenged Gsdme^−/− mice. However, GSDME knockdown did not lead to the further increase of mRNA and secretion of TNF-α and IL-6 in UVB-challenged keratinocytes. Skin damage was not aggravated in UVB-challenged Gsdme cKO mice. Neutrophils depletion alleviated UVB-caused skin damage in WT mice and Gsdme^−/− mice, and eliminated its aggravation in Gsdme^−/− mice. This study demonstrates that GSDME plays a restrictive role in UVB-induced skin damage through inhibiting excessive recruitment and activation of neutrophils in the immune microenvironment in UVB-caused skin inflammation. However, keratinocytes might not contribute to this restrictive function.Subject terms: Cell death, Cell death and immune response, Inflammation     

Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15 ± 2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8 ± 1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in tumors induced in these two murine models suggesting the specificity of Notch pathway in this regard. These data provide a novel role of ODC in augmenting tumorigenesis via negatively regulated Notch-mediated expansion of stem cell compartment.     

Stathmin Is Dispensable for Tumor Onset in Mice

The microtubule-destabilizing protein stathmin is highly expressed in several types of tumor, thus deserving the name of oncoprotein 18. High levels of stathmin expression and/or activity favor the metastatic spreading and mark the most aggressive tumors, thus representing a realistic marker of poor prognosis. Stathmin is a downstream target of many signaling pathways, including Ras-MAPK, PI3K and p53, involved in both tumor onset and progression. We thus hypothesized that stathmin could also play a role during the early stages of tumorigenesis, an issue completely unexplored. In order to establish whether stathmin expression is necessary for tumor initiation, we challenged wild type (WT), stathmin heterozygous and stathmin knock-out (KO) mice with different carcinogens. Using well-defined mouse models of carcinogenesis of skin, bladder and muscle by the means of 7,12-dimethylbenz[α]antracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and 3-methylcholanthrylene (3MC) treatments, respectively, we demonstrated that knock-out of stathmin has no impact on the onset of cancer in mice. No significant difference was noticed either when the Ras oncogene was mutated (skin carcinogenesis model) or when the p53 pathway was inactivated (bladder carcinomas and fibrosarcomas). Finally, we concomitantly impinged on p53 and Ras pathways, by generating WT and stathmin KO mouse embryo fibroblasts transformed with papilloma virus large T antigen (LgTAg) plus the K-Ras^G12V oncogene. In vivo growth of xenografts from these transformed fibroblasts did not highlight any significant difference depending on the presence or absence of stathmin. Overall, our work demonstrates that stathmin expression is dispensable for tumor onset, at least in mice, thus making stathmin a virtually exclusive marker of aggressive disease and a promising therapeutic target for advanced cancers.     

Nod-Like Receptor Protein-3 Inflammasome Plays an Important Role during Early Stages of Wound Healing

The Nod-like receptor protein (NLRP)-3 inflammasome/IL-1β pathway is involved in the pathogenesis of various inflammatory skin diseases, but its biological role in wound healing remains to be elucidated. Since inflammation is typically thought to impede healing, we hypothesized that loss of NLRP-3 activity would result in a downregulated inflammatory response and accelerated wound healing. NLRP-3 null mice, caspase-1 null mice and C57Bl/6 wild type control mice (WT) received four 8 mm excisional cutaneous wounds; inflammation and healing were assessed during the early stage of wound healing. Consistent with our hypothesis, wounds from NLRP-3 null and caspase-1 null mice contained lower levels of the pro-inflammatory cytokines IL-1β and TNF-α compared to WT mice and had reduced neutrophil and macrophage accumulation. Contrary to our hypothesis, re-epithelialization, granulation tissue formation, and angiogenesis were delayed in NLRP-3 null mice and caspase-1 null mice compared to WT mice, indicating that NLRP-3 signaling is important for early events in wound healing. Topical treatment of excisional wounds with recombinant IL-1β partially restored granulation tissue formation in wounds of NLRP-3 null mice, confirming the importance of NLRP-3-dependent IL-1β production during early wound healing. Despite the improvement in healing, angiogenesis and levels of the pro-angiogenic growth factor VEGF were further reduced in IL-1β treated wounds, suggesting that IL-1β has a negative effect on angiogenesis and that NLRP-3 promotes angiogenesis in an IL-1β-independent manner. These findings indicate that the NLRP-3 inflammasome contributes to the early inflammatory phase following skin wounding and is important for efficient healing.     

Manipulation of Catalase Levels Produces Altered Photosynthesis in Transgenic Tobacco Plants

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalytic form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO₂ compensation point (Γ) at a leaf temperature of 38°C. A significant linear increase was observed in Γ with decreasing catalase (at 50% lower catalase activity Γ increased 39%). There was a significant temperature-dependent linear decrease in Γ in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase Γ decreased 17%). At 29°C, Γ also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO₂ were significantly reduced with increasing catalase activities at 38°C, indicating that the stoichiometry of photorespiratory CO₂ formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.     

Quantitative scanning electron microscopy of solitary chemoreceptor cells in cyprinids and other teleosts

Synopsis Solitary chemosensory cells (SCC) occur in the epidermis of many lower, aquatic vertebrates. By scanning electron microscopy, SCC apices were counted and density distributions estimated along various transects at the head and body of 12 species of teleost fishes, 7 cyprinids, 2 perciforms, 2 catfish and 1 characinid. In contrast to taste buds (TB), the distribution of SCCs is relatively even, with slightly higher densities at the forehead and along the dorsal trunk. In most species 1000 to 1500 SCC apices per mm² of skin were counted. Considerably higher densities occur in halos around free neuromasts. Depending on fish size and apex density, the epidermis of individuals may contain millions of SCCs. SCCs are considerably more abundant in individual fish than TB sensory cells. Highest average SCC densities (2000–4000 per mm²) were found in the cyprinids, roach, nase, chub and bream. Lowest densities (250 per mm²) occurred in the neon tetra. No correlations could be found between SCC densities and TB densities or relative size of the brain stem facial lobe, supporting the view of different functions and biological roles of the SCC and the TB systems. Whether teleost SCCs generally respond to mucoid substances, as in the case of the rocklings, remains an open question.     

Activation of Myocardial Phosphoinositide-3-Kinase p110α Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p<0.01) and 50% survived >30 days (p<0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110α expression. Cardiac high mobility group box–1 (HMGB-1) translocation was also inhibited by caPI3K p110α expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110α signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.     

A Potential Link between the C5a Receptor 1 and the β1-Adrenoreceptor in the Mouse Heart

Purpose

Inflammation may contribute to the pathogenesis of specific cardiovascular diseases, but it is uncertain if mediators released during the inflammatory process will affect the continued efficacy of drugs used to treat clinical signs of the cardiac disease. We investigated the role of the complement 5a receptor 1 (C5aR1/CD88) in the cardiac response to inflammation or atenolol, and the effect of C5aR1 deletion in control of baseline heart rate in an anesthetized mouse model.

Methods

An initial study showed that PMX53, an antagonist of C5aR1 in normal C57BL6/J (wild type, WT) mice reduced heart rate (HR) and appeared to have a protective effect on the heart following induced sepsis. C5aR1 knockout (CD88-/-) mice had a lower HR than wild type mice, even during sham surgery. A model to assess heart rate variability (HRV) in anesthetized mice was developed to assess the effects of inhibiting the β₁-adrenoreceptor (β₁-AR) in a randomized crossover study design.

Results

HR and LF Norm were constitutively lower and SDNN and HF Norm constitutively higher in the CD88-/- compared with WT mice (P< 0.001 for all outcomes). Administration of atenolol (2.5 mg/kg) reduced the HR and increased HRV (P< 0.05, respectively) in the wild type but not in the CD88-/- mice. There was no shift of the sympathovagal balance post-atenolol in either strains of mice (P> 0.05), except for the reduced LF/HF (Lower frequency/High frequency) ratio (P< 0.05) at 60 min post-atenolol, suggesting increased parasympathetic tone of the heart due to the effect of atenolol administration. The HR of the WT mice were lower post atenolol compared to the CD88-/- mice (P = 0.001) but the HRV of CD88-/- mice were significantly increased (P< 0.05), compared with WT mice.

Conclusion

Knockout of the C5aR1 attenuated the effect of β₁-AR in the heart, suggesting an association between the β₁-AR and C5aR1, although further investigation is required to determine if this is a direct or causal association.     

Low Na,High K Diet and the Role of Aldosterone in BK-Mediated K Excretion

A low Na, high K diet (LNaHK) is associated with a low rate of cardiovascular (CV) disease in many societies. Part of the benefit of LNaHK relies on its diuretic effects; however, the role of aldosterone (aldo) in the diuresis is not understood. LNaHK mice exhibit an increase in renal K secretion that is dependent on the large, Ca-activated K channel, (BK-α with accessory BK-β4; BK-α/β4). We hypothesized that aldo causes an osmotic diuresis by increasing BK-α/β4-mediated K secretion in LNaHK mice. We found that the plasma aldo concentration (P[aldo]) was elevated by 10-fold in LNaHK mice compared with control diet (Con) mice. We subjected LNaHK mice to either sham surgery (sham), adrenalectomy (ADX) with low aldo replacement (ADX-LA), or ADX with high aldo replacement (ADX-HA). Compared to sham, the urinary flow, K excretion rate, transtubular K gradient (TTKG), and BK-α and BK-β4 expressions, were decreased in ADX-LA, but not different in ADX-HA. BK-β4 knockout (β4KO) and WT mice exhibited similar K clearance and TTKG in the ADX-LA groups; however, in sham and ADX-HA, the K clearance and TTKG of β4KO were less than WT. In response to amiloride treatment, the osmolar clearance was increased in WT Con, decreased in WT LNaHK, and unchanged in β4KO LNaHK. These data show that the high P[aldo] of LNaHK mice is necessary to generate a high rate of BK-α/β4-mediated K secretion, which creates an osmotic diuresis that may contribute to a reduction in CV disease.