Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.

Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause strokes and seizures in younger individuals. CCMs arise from endothelial cell loss of KRIT1, CCM2 or PDCD10, non-homologous proteins that form an adaptor complex. How disruption of the CCM complex results in disease remains controversial, with numerous signalling pathways (including Rho, SMAD and Wnt/ß-catenin) and processes such as endothelial-mesenchymal transition (EndMT) proposed to have causal roles. CCM2 binds to MEKK3 (refs 7, 8, 9, 10, 11), and we have recently shown that CCM complex regulation of MEKK3 is essential during vertebrate heart development. Here we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we show that expression of the MEKK3 target genes Klf2 and Klf4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. By contrast, we find no evidence of EndMT or increased SMAD or Wnt signalling during early CCM formation. Endothelial-specific loss of Map3k3 (also known as Mekk3), Klf2 or Klf4 markedly prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we show that endothelial expression of KLF2 and KLF4 is increased in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates the MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signalling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics.

Anti-inflammatory γ- and δ-tocotrienols improve cardiovascular, liver and metabolic function in diet-induced obese rats.

PURPOSE: This study tested the hypothesis that γ- and δ-tocotrienols are more effective than α-tocotrienol and α-tocopherol in attenuating the signs of diet-induced metabolic syndrome in rats. METHODS: Five groups of rats were fed a corn starch-rich (C) diet containing 68 % carbohydrates as polysaccharides, while the other five groups were fed a diet (H) high in simple carbohydrates (fructose and sucrose in food, 25 % fructose in drinking water, total 68 %) and fats (beef tallow, total 24 %) for 16 weeks. Separate groups from each diet were supplemented with either α-, γ-, δ-tocotrienol or α-tocopherol (85 mg/kg/day) for the final 8 of the 16 weeks. RESULTS: H rats developed visceral obesity, hypertension, insulin resistance, cardiovascular remodelling and fatty liver. α-Tocopherol, α-, γ- and δ-tocotrienols reduced collagen deposition and inflammatory cell infiltration in the heart. Only γ- and δ-tocotrienols improved cardiovascular function and normalised systolic blood pressure compared to H rats. Further, δ-tocotrienol improved glucose tolerance, insulin sensitivity, lipid profile and abdominal adiposity. In the liver, these interventions reduced lipid accumulation, inflammatory infiltrates and plasma liver enzyme activities. Tocotrienols were measured in heart, liver and adipose tissue showing that chronic oral dosage delivered tocotrienols to these organs despite low or no detection of tocotrienols in plasma. CONCLUSION: In rats, δ-tocotrienol improved inflammation, heart structure and function, and liver structure and function, while γ-tocotrienol produced more modest improvements, with minimal changes with α-tocotrienol and α-tocopherol. The most important mechanism of action is likely to be reduction in organ inflammation.

Micro-CT Imaging Reveals Mekk3 Heterozygosity Prevents Cerebral Cavernous Malformations in Ccm2-Deficient Mice.

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5µm (leading to a minimum feature size of approximately 25µm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

Deletion of Wntless in myeloid cells exacerbates liver fibrosis and the ductular reaction in chronic liver injury.

BACKGROUND: Macrophages play critical roles in liver regeneration, fibrosis development and resolution. They are among the first responders to liver injury and are implicated in orchestrating the fibrogenic response via multiple mechanisms. Macrophages are also intimately associated with the activated hepatic progenitor cell (HPC) niche or ductular reaction that develops in parallel with fibrosis. Among the many macrophage-derived mediators implicated in liver disease progression, a key role for macrophage-derived Wnt proteins in driving pro-regenerative HPC activation towards a hepatocellular fate has been suggested. Wnt proteins, in general, however, have been associated with both pro- and anti-fibrogenic activities in the liver and other organs. We investigated the role of macrophage-derived Wnt proteins in fibrogenesis and HPC activation in murine models of chronic liver disease by conditionally deleting Wntless expression, which encodes a chaperone essential for Wnt protein secretion, in LysM-Cre-expressing myeloid cells (LysM-Wls mice). RESULTS: Fibrosis and HPC activation were exacerbated in LysM-Wls mice compared to littermate controls, in the absence of an apparent increase in myofibroblast activation or interstitial collagen mRNA expression, in both the TAA and CDE models of chronic liver disease. Increased Epcam mRNA levels paralleled the increased HPC activation and more mature ductular reactions, in LysM-Wls mice. Increased Epcam expression in LysM-Wls HPC was also observed, consistent with a more cholangiocytic phenotype. No differences in the mRNA expression levels of key pro-inflammatory and pro-fibrotic cytokines or the macrophage-derived HPC mitogen, Tweak, were observed. LysM-Wls mice exhibited increased expression of Timp1, encoding the key Mmp inhibitor Timp1 that blocks interstitial collagen degradation, and, in the TAA model, reduced expression of the anti-fibrotic matrix metalloproteinases, Mmp12 and Mmp13, suggesting a role for macrophage-derived Wnt proteins in restraining fibrogenesis during ongoing liver injury. CONCLUSION: In summary, these data suggest that macrophage-derived Wnt proteins possess anti-fibrogenic potential in chronic liver disease, which may be able to be manipulated for therapeutic benefit.

Tocotrienols reverse cardiovascular, metabolic and liver changes in high carbohydrate, high fat diet-fed rats.

Tocotrienols have been reported to improve lipid profiles, reduce atherosclerotic lesions, decrease blood glucose and glycated haemoglobin concentrations, normalise blood pressure in vivo and inhibit adipogenesis in vitro, yet their role in the metabolic syndrome has not been investigated. In this study, we investigated the effects of palm tocotrienol-rich fraction (TRF) on high carbohydrate, high fat diet-induced metabolic, cardiovascular and liver dysfunction in rats. Rats fed a high carbohydrate, high fat diet for 16 weeks developed abdominal obesity, hypertension, impaired glucose and insulin tolerance with increased ventricular stiffness, lower systolic function and reduced liver function. TRF treatment improved ventricular function, attenuated cardiac stiffness and hypertension, and improved glucose and insulin tolerance, with reduced left ventricular collagen deposition and inflammatory cell infiltration. TRF improved liver structure and function with reduced plasma liver enzymes, inflammatory cell infiltration, fat vacuoles and balloon hepatocytes. TRF reduced plasma free fatty acid and triglyceride concentrations but only omental fat deposition was decreased in the abdomen. These results suggest that tocotrienols protect the heart and liver, and improve plasma glucose and lipid profiles with minimal changes in abdominal obesity in this model of human metabolic syndrome.

Caffeine attenuates metabolic syndrome in diet-induced obese rats.

OBJECTIVE: Caffeine is a constituent of many non-alcoholic beverages. Pharmacological actions of caffeine include the antagonism of adenosine receptors and the inhibition of phosphodiesterase activity. The A1 adenosine receptors present on adipocytes are involved in the control of fatty acid uptake and lipolysis. In this study, the effects of caffeine were characterized in a diet-induced metabolic syndrome in rats. METHODS: Rats were given a high-carbohydrate, high-fat diet (mainly containing fructose and beef tallow) for 16 wk. The control rats were given a corn starch diet. Treatment groups were given caffeine 0.5 g/kg of food for the last 8 wk of the 16-wk protocol. The structure and function of the heart and the liver were investigated in addition to the metabolic parameters including the plasma lipid components. RESULTS: The high-carbohydrate, high-fat diet induced symptoms of metabolic syndrome, including obesity, dyslipidemia, impaired glucose tolerance, decreased insulin sensitivity, and increased systolic blood pressure, associated with the development of cardiovascular remodeling and non-alcoholic steatohepatitis. The treatment with caffeine in the rats fed the high-carbohydrate, high-fat diet decreased body fat and systolic blood pressure, improved glucose tolerance and insulin sensitivity, and attenuated cardiovascular and hepatic abnormalities, although the plasma lipid concentrations were further increased. CONCLUSION: Decreased total body fat, concurrent with increased plasma lipid concentrations, reflects the lipolytic effects of caffeine in adipocytes, likely owing to the caffeine antagonism of A1 adenosine receptors on adipocytes.