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Studies have suggested a link between the transforming growth factor beta 1 (TGF-β1) signaling cascade and the stress-inducible activating transcription factor 3 (ATF3). We have demonstrated that triglyceride-rich lipoproteins (TGRL) lipolysis products activate MAP kinase stress associated JNK/c-Jun pathways resulting in up-regulation of ATF3, pro-inflammatory genes and induction of apoptosis in human aortic endothelial cells. Here we demonstrate increased release of active TGF-β at 15 min, phosphorylation of Smad2 and translocation of co-Smad4 from cytosol to nucleus after a 1.5 h treatment with lipolysis products. Activation and translocation of Smad2 and 4 was blocked by addition of SB431542 (10 μM), a specific inhibitor of TGF-β-activin receptor ALKs 4, 5, 7. Both ALK receptor inhibition and anti TGF-β1 antibody prevented lipolysis product induced up-regulation of ATF3 mRNA and protein. ALK inhibition prevented lipolysis product-induced nuclear accumulation of ATF3. ALKs 4, 5, 7 inhibition also prevented phosphorylation of c-Jun and TGRL lipolysis product-induced p53 and caspase-3 protein expression. These findings demonstrate that TGRL lipolysis products cause release of active TGF-β and lipolysis product-induced apoptosis is dependent on TGF-β signaling. Furthermore, signaling through the stress associated JNK/c-Jun pathway is dependent on TGF-β signaling suggesting that TGF-β signaling is necessary for nuclear accumulation of the ATF3/cJun transcription complex and induction of pro-inflammatory responses.

Products generated from lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TGRL) are reported to increase endothelial layer permeability. We hypothesize that these increases in permeability result from the active rearrangement and dissolution of the junctional barrier in human aortic endothelial cells, as well as induction of the apoptotic cascade. Human aortic endothelial cells were treated with TGRL lipolysis products generated from coincubation of human TGRL plus lipoprotein lipase. Measurement of transendothelial electrical resistance demonstrated a time-dependent decrease in endothelial barrier function in response to TGRL lipolysis products. Immunofluorescent localization of zonula occludens-1 (ZO-1) showed radial rearrangement along cell borders after 1.5 h of treatment with lipolysis products. A concurrent redistribution of F-actin from the cell body to the cell margins was observed via rhodamine phalloidin staining. Immunofluorescent imaging for occludin and vascular endothelial cadherin showed that these proteins relocalize as well, although these changes are less prominent than for ZO-1. Western analysis of cells exposed to lipolysis products for 3 h revealed the fragmentation of ZO-1, a reduction in occludin, and no change of vascular endothelial cadherin. Lipolysis products also increased caspase-3 activity and induced nuclear fragmentation. Treatments did not cause oncosis in cells at any point during the incubation. These results demonstrate that TGRL lipolysis products play an important role in the regulation of endothelial permeability, the organization of the actin cytoskeleton, the localization and expression of junctional proteins, especially ZO-1, and the induction of apoptosis.

The overall goal of this project was to examine the interactions of hyperglycemia and loss of ovarian hormones on the artery wall in a type I diabetic mouse model. Intact or ovariectomized (OVX) female BALB/C mice were fed a high-cholesterol diet. Half the animals were treated with steptozotocin to induce insulin-deficient diabetes mellitus, generating four treatment groups: control, intact; control, ovariectomized; diabetic, intact; diabetic, ovariectomized (DOVX). We examined arterial structure and function and found that 1) diabetes and ovariectomy additively increased endothelial layer permeability, 2) arterial stiffening was increased in DOVX, 3) DOVX synergistically increased atheroma formation, and 4) ultrastructural evaluation revealed that the basal lamina was often multilayered and formed convoluted aggregates separating endothelium from the internal elastic lamina in diabetic, but not control arteries or arteries from OVX mice. Endothelium overlying these regions formed thin cytoplasmic extensions between these aggregates and was often separated from the basal lamina by electron lucent spaces. Our studies showed that diabetes and loss of ovarian function have additive and synergistic effects to worsen arterial pathophysiology by disrupting the arterial endothelial layer with increased permeability and increased atheroma formation.