Identification of PKCalpha isoform-specific effects in cardiac myocytes using antisense phosphorothioate oligonucleotides

Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in multiple cellular processes. In the heart, PKC signaling is involved in hypertrophic agonist-induced gene expression and hypertrophic growth. To investigate the specific function of PKC signaling in regulating cardiomyocyte growth, we used antisense oligonucleotides to inhibit PKC alpha, the major isozyme present in the neonatal heart. Transfection of cultured neonatal cardiomyocytes with antisense PKCalpha oligonucleotides resulted in a marked reduction in both PKCalpha mRNA and protein levels. PKCalpha antisense treatment also reduced phenylephrine (PE)-induced PKC activity and perinuclear translocation of PKCalpha. Antisense inhibition of PKCalpha led to reduction of PE-induced increase in skeletal alpha-actin mRNA levels and atrial natriuretic peptide (ANP) secretion but had no significant effects on PE-induced beta-myosin heavy chain, ANP, or B-type natriuretic peptide (BNP) gene expression. On the other hand, antisense PKCalpha treatment attenuated endothelin-1-induced increase in ANP and BNP peptide secretion, whereas endothelin-1-induced gene expression of ANP and BNP remained unchanged. The hypertrophic agonist-induced growth of cardiomyocytes, characterized by increased [(3)H]leucine incorporation, was not affected with antisense PKCalpha treatment. Furthermore, we found that PE-induced increase in extracellular signal-regulated kinase (ERK) activity was partially inhibited by antisense PKCalpha treatment, implicating ERK as a downstream mediator for PKCalpha signaling. These results indicate that PKCalpha isozyme is involved in hypertrophic signaling in cardiomyocytes and provide novel strategies for future studies to identify other cellular targets controlled selectively by PKCalpha or other PKC isozymes.     

Cytosolic Phospholipase A2α Protects against Ischemia/Reperfusion Injury in the Heart

Studies with sPLA₂ Group X, and cPLA₂α gene‐targeted mice suggest that absence of sPLA₂ Group X results in protection from ischemia/reperfusion (I/R) injury in the heart, and absence of cPLA₂α Group IV is protective in the brain. Although latter studies might suggest a similar deleterious role for cPLA₂α in I/R injury in the heart, the pathophysiology of stroke is intricately related to excitotoxicity and cannot necessarily be extrapolated to the heart. We report here that unlike findings in the brain, cPLA₂α^(−/−) mice have exaggerated injury following I/R in vivo. In contrast, there is no difference in injury induced by simulated ischemia in cardiomyocytes isolated from cPLA₂α^(−/−) versus cPLA₂α^(+/+) mice. This suggests that cPLA₂α does not have an important cardiomyocyte autonomous effect on ischemic injury. Prostaglandin E₂ (PGE₂) levels are significantly reduced in the hearts of the cPLA₂α^(−/−) mice, and the enhanced injury is ameliorated by treatment with the PGE analog, misoprostol. We demonstrate that cPLA₂α is cardioprotective in vivo, and this is likely via cPLA₂α‐mediated production of cardioprotective eicosanoids. These studies are the first to identify a protective role for cPLA₂ in I/R injury in any organ and raise concerns over long‐term inhibition of cPLA₂. Clin Trans Sci 2011; Volume 4: 236–242     

Expression of human macrophage metalloelastase (MMP-12) by tumor cells in skin cancer

Matrix metalloproteinases play an essential role in tumor growth and invasion. Different matrix metalloproteinases are often expressed in cancers with distinct patterns. To investigate the role of human macrophage metalloelastase (MMP-12) in epidermal tumors, we studied human macrophage metalloelastase mRNA and protein expression in malignant squamous cell and basal cell carcinomas, and in premalignant Bowen's disease. Human macrophage metalloelastase was detected in 11 of 17 squamous cell carcinomas in epithelial cancer cells, whereas macrophages were positive in 15 of 17 samples. In basal cell carcinomas, human macrophage metalloelastase was more often found in macrophages (seven of 19) than in cancer cells (four of 19). Human macrophage metalloelastase mRNA was also detected in three cell lines derived from squamous cell carcinomas of the head and neck and in transformed HaCaT cells, whereas premalignant tumors and primary keratinocytes were negative for human macrophage metalloelastase mRNA. Western analysis revealed human macrophage metalloelastase protein in squamous cell carcinoma cells. Our results show that human macrophage metalloelastase can be expressed in vivo and in vitro by transformed epithelial cells and indicate that the level of human macrophage metalloelastase expression correlates with epithelial dedifferentiation and histologic aggressiveness.     

Associations Between Maternal Prenatal C-Reactive Protein and Risk Factors for Psychosis in Adolescent Offspring: Findings From the Northern Finland Birth Cohort 1986

Prenatal infection is associated with brain structural and functional abnormalities and may increase the risk for psychosis through a direct effect on neurodevelopment. Various infections may exert their effect through a proinflammatory immune response but studies of prenatal maternal inflammatory markers and offspring neurodevelopment are scarce. Using the longitudinal Northern Finland Birth Cohort 1986 study, we examined the associations of maternal prenatal C-reactive protein (CRP) levels with psychosis risk factors in adolescent offspring. CRP was measured in maternal sera collected in pregnancy. In offspring, school performance was measured at age 7 years, while school performance, psychotic experiences, and cannabis use were measured at age 16 years. We tested associations of CRP with offspring measures using regression analysis controlling for offspring sex, maternal education level, and prenatal maternal body mass index, smoking and alcohol use in pregnancy, place of birth, maternal psychiatric admission, paternal psychiatric admission, mothers age at birth, and gestational week of CRP sample. We also tested if adolescent cannabis use mediated the associations between maternal CRP and offspring outcomes. Controlling for covariates, maternal CRP was associated with academic performance at age 16 years (beta = .062, 95% CI = 0.036–0.088), but not with possible psychotic experiences at 16 years (odds ratio [OR] = 1.09, 95% CI = 0.96–1.24). Maternal CRP was also associated with adolescent cannabis use (OR = 1.24, 95% CI = 1.07–1.43). These findings suggest that prenatal inflammation may influence later mental illness risk by affecting neurodevelopment and also indirectly by increasing the risk of exposure to cannabis.     

Fracture of titanium plates used for mandibular reconstruction following ablative tumor surgery

PURPOSE: The purpose of this study was to identify reasons for fracture of titanium mandibular reconstruction plates, when used to bridge lateral mandibular defects after ablative tumor surgery. MATERIALS AND METHODS: Sixteen titanium reconstruction plates from sheep mandibles were examined to identify reasons for the plate fractures. The broken plates and the seemingly unbroken plates were examined separately. The plates were removed from the mandibular bone and inspected by dye penetrant examination, metallography, optical microscope, scanning electron microscope, and energy dispersive X-ray spectrometer. Furthermore, axial load fatigue tests were performed in two different environments, air and physiologic salt solution, 0.9% NaCl, to compare titanium behavior in air and the human body. RESULTS: The site of crack initiation was the inner curvature of the reconstruction plate, and the cracks initiated as a result of stress concentration in the shoulder fillet of the plate. The cracks grew in a cyclic manner under masticatory loading of the mandible and the plate. The plate fracture occurred by means of fatigue. The corrosive environment did not affect the failure of the titanium plate, and the fracture was not caused by hydrogen embrittlement. The results revealed that the fatigue properties of the plates may have been impaired by the residual stresses generated in plate bending. CONCLUSIONS: Adjustive bending of the plates, in the surgical operation, may thus be an important cause of fracture of the reconstruction plates, because of generated residual stresses, which affect the mean stress in fatigue loading. To make the plates function without failure the plates should match closely with the three-dimensional shape of the mandible, to avoid any bending in the operative phase.     

Cytosolic phospholipase A(2)α protects against ischemia/reperfusion injury in the heart

Studies with sPLA(2) Group X, and cPLA(2) α gene-targeted mice suggest that absence of sPLA(2) Group X results in protection from ischemia/reperfusion (I/R) injury in the heart, and absence of cPLA(2) α Group IV is protective in the brain. Although latter studies might suggest a similar deleterious role for cPLA(2) α in I/R injury in the heart, the pathophysiology of stroke is intricately related to excitotoxicity and cannot necessarily be extrapolated to the heart. We report here that unlike findings in the brain, cPLA(2) α((-/-)) mice have exaggerated injury following I/R in vivo. In contrast, there is no difference in injury induced by simulated ischemia in cardiomyocytes isolated from cPLA(2) α((-/-)) versus cPLA(2) α((+/+)) mice. This suggests that cPLA(2) α does not have an important cardiomyocyte autonomous effect on ischemic injury. Prostaglandin E(2) (PGE(2) ) levels are significantly reduced in the hearts of the cPLA(2) α((-/-)) mice, and the enhanced injury is ameliorated by treatment with the PGE analog, misoprostol. We demonstrate that cPLA(2) α is cardioprotective in vivo, and this is likely via cPLA(2) α-mediated production of cardioprotective eicosanoids. These studies are the first to identify a protective role for cPLA(2) in I/R injury in any organ and raise concerns over long-term inhibition of cPLA(2).