Shear stress regulation of Krüppel-like factor 2 expression is flow pattern-specific

Flow patterns in blood vessels contribute to focal distribution of atherosclerosis; the underlying mechanotransduction pathways remain to be investigated. We demonstrate that different flow patterns elicit distinct responses of Krüppel-like factor-2 (KLF2) in endothelial cells (ECs) in vitro and in vivo. While pulsatile flow with a significant forward direction induced sustained expression of KLF2 in cultured ECs, oscillatory flow with little forward direction caused prolonged suppression after a transient induction. The suppressive effect of oscillatory flow was Src-dependent. Immunohistochemical studies on ECs at arterial branch points revealed that KLF2 protein levels were related to local hemodynamics. Such flow-associated expression patterns were also demonstrated in a rat aortic restenosis model. Inhibition of KLF2 with siRNA sensitized ECs to oxidized LDL-induced apoptosis, indicating a protective role of KLF2. In conclusion, differential regulation of KLF2 may mediate the distinct vascular effects induced by various patterns of shear stress.     

Integrin α7β1 is a redox-regulated target of hydrogen peroxide in vascular smooth muscle cell adhesion

Upon adhesion to laminin-111, aortic smooth muscle cells initially form membrane protrusions with an average diameter of 2.9μm. We identified these protrusions also as subcellular areas of increased redox potential and protein oxidation by detecting cysteine sulfenic acid groups with dimedone. Hence, we termed these areas oxidative hot spots. They are spatially and temporally transient during an early stage of adhesion and depend on the activity of the H(2)O(2)-generating NADPH oxidase 4. Presumably located on cellular protrusions, integrin α7β1 mediates adhesion and migration of vascular smooth muscle cells to laminins of their surrounding basement membrane. Using protein chemistry and mass spectrometry, two specific oxidation sites within the integrin α7 subunit were identified: one located in its genu region and another within its calf 2 domain. Upon H(2)O(2) treatment, two cysteine residues are oxidized thereby unlocking a disulfide bridge. The genu region is a hinge, around which the integrin domains pivot between a bent/inactive and an upright/active conformation. Also, cysteine oxidation within the calf 2 domain permits conformational changes related to integrin activation. H(2)O(2) treatment of α7β1 integrin in concentrations of up to 100μM increases integrin binding activity to laminin-111, suggesting a physiological redox regulation of α7β1 integrin.     

Krüppel-like factor 8 (KLF8) is expressed in gliomas of different WHO grades and is essential for tumor cell proliferation

Krüppel-like factor 8 (KLF8) has only recently been identified to be involved in tumor cell proliferation and invasion of several different tumor entities like renal cell carcinoma, hepatocellular carcinoma and breast cancer. In the present study, we show for the first time the expression of KLF8 in gliomas of different WHO grades and its functional impact on glioma cell proliferation. In order to get information about KLF8-mRNA regulation qPCR was performed and did not reveal any significant difference in samples (n = 10 each) of non-neoplastic brain (NNB), low-grade gliomas (LGG, WHO°II) and glioblastomas (GBM, WHO°IV). Immunohistochemistry of tissue samples (n = 7 LGG, 11 AA and 12 GBM) did not show any significant difference in the fraction of KLF8-immunopositive cells of all analyzed cells in LGG (87%), AA (80%) or GBM (89%). Tissue samples from cerebral breast cancer metastasis, meningiomas but also non-neoplastic brain demonstrated comparable relative cell counts as well. Moreover, there was no correlation between KLF8 expression and the expression pattern of the assumed proliferation marker Ki67, which showed high variability between different tumor grade (9% (LGG), 6% (AA) and 15% (GBM) of Ki67-immunopositive cells). Densitometric analysis of Western blotting revealed that the relative amount of KLF8-protein did also not differ between the highly aggressive and proliferative GBM (1.05) compared to LGG (0.93; p<0.05, studens t-test). As demonstrated for some other non-glial cancer entities, KLF8-knockdown by shRNA in U87-MG cells confirmed its functional relevance, leading to an almost complete loss of tumor cell proliferation. Selective blocking of KLF8 might represent a novel anti-proliferative treatment strategy for malignant gliomas. Yet, its simultaneous expression in non-proliferating tissues could hamper this approach.