Abdominal aortic aneurysms (AAAs) are a major cause of morbidity and mortality in the United States today. We employed a model for AAA development using apolipoprotein E knock out mice fed a high-fat diet and treated with ANG II and β-aminopropionitrile (β-APN) for 4 wk. ANG II induces hypertension and atherosclerotic disease, whereas β-APN inhibits the activity of the lysyl oxidase/ lysyl oxidase-like protein (LOX/LOXL) family members. LOX/LOXL family members crosslink collagen and elastin in the extracellular matrix and therefore contribute to the integrity and stabilization of a healthy vessel wall. In this model, cotreatment with ANG II and β-APN caused a 90% AAA incidence and increased atherosclerotic lesion formation from less than 5% to greater than 25% after 4 wk. In more atheroprotected mouse strains (C57BL/6 and BalbC), cotreatment with ANG II and β-APN caused 50% and 40% AAA incidence, respectively. These data demonstrate the importance of LOX/LOXL to the stability of the vessel wall. Therapeutic strategies to overexpress LOX/LOXL enzymes or to support the crosslinking of soluble matrix proteins in a polymeric scaffold are a promising opportunity to achieve stabilization of AAAs.

Objective Osteopontin (OPN) is a multifunctional protein found in abundance in atherosclerotic plaques. Angiotensin II (Ang II) promotes atherosclerosis by inducing adhesion and migration of vascular smooth muscle cells (VSMCs). MicroRNAs (miRNAs) are critical regulators of protein expression. However, the relationship between Ang II, miRNAs and OPN has yet to be fully explored. Methods and results Using cultured VSMCs, we found that Ang II increased cellular OPN protein expression 4 h after treatment by 420 ± 54% (p < 0.03) in a translation dependent manner. Sequence analysis revealed a putative binding site for mir181a and raised the possibility that miR181a is a potential regulatory mechanism for OPN expression. We demonstrated that Ang II decreased miR181a expression by 52 ± 7% (p < 0.0001) and overexpressing miR181a inhibited Ang II induced increases in OPN protein expression by 69 ± 9% (p < 0.05). Furthermore, we demonstrated that miR181a is functionally important in that overexpression of miR181a inhibited VSMCs adhesion to collagen in response to Ang II as compared to controls by 36 ± 4%. (p < 0.05) Conclusions These results demonstrate that miR181a regulates OPN expression and that altering miR181a expression may be a novel therapeutic approach to modulate OPN protein expression.

Background: Osteopontin expression is increased in numerous diseases with underlying increases in H 2 O 2 . Results: H 2 O 2 -dependent osteopontin expression is biphasic. Early increases occur through translation via redox-dependent 4EPB-1, whereas late increases require NF-B-and AP-1-dependent transcription. Conclusion: H 2 O 2 -induced osteopontin expression is both translational and transcriptional. Significance: Understanding how osteopontin is regulated is critical for targeting this inflammatory protein in H 2 O 2 -dependent pathologies.

The neuroprotective effects of progesterone after ischemic stroke have been established, but the role of progesterone in promoting cerebrovascular repair remains under-explored. Male Sprague–Dawley rats underwent transient middle cerebral artery occlusion (tMCAO) for 90 min followed by reperfusion for 3 days. Progesterone (8 mg/kg/day) was administered intraperitoneally at 1 hour after initial occlusion followed by subcutaneous injections at 6, 24 and 48 hours post-occlusion. Rats were euthanized after 72 hours and brain endothelial cell density and macrophage infiltration were evaluated within the cerebral cortex. We also assessed progesterone’s ability to induce macrophage migration towards hypoxic/reoxygenated cultured endothelial cells. We found that progesterone treatment post-tMCAO protects ischemic endothelial cells from macrophage infiltration. We further demonstrate that infiltration of monocytes/macrophages can be induced by potent chemotactic factors such as monocyte chemoattractant protein-1 (MCP-1) and the chemokine ligand 1 (CXCL1), secreted by hypoxic/reoxygenated endothelial cells. Progesterone blunts secretion of MCP-1 and CXCL1 from endothelial cells after hypoxia/reoxygenation injury and decreases leukocyte infiltration. The treatment protects ischemic endothelial cells from macrophage infiltration and thus preserves vascularization after ischemic injury.

The occlusion of blood vessels in the setting of cardiovascular disease leads to ischemia, initiating processes that promote neovascularization to restore blood flow and preserve tissue function. Our in vivo studies show that Osteopontin (OPN) is a critical mediator of post-ischemic neovascularization and that ischemia-induced increases in OPN expression are H 2 O 2 -dependent. However, the mechanisms by which H 2 O 2 increases OPN expression are poorly defined. To determine if H 2 O 2 mediates transcriptional, post-transcriptional, and/or translational regulation of OPN expression in vitro, we used rat aortic smooth muscle cells as an in vitro system and stimulated with H 2 O 2 . Dose response studies showed OPN expression increased with 50 μM H 2 O 2 (51.9%±2.2, p<0.05). Using 50 μM H 2 O 2 , we performed time courses and measured OPN mRNA by qRT-PCR and protein by Western blot. OPN mRNA levels significantly increased in response to H 2 O 2 at 8 (70.4%±5.7, p<0.05) and 18 hours (120.2%±5.2, p<0.005). Interestingly, the increases in OPN protein expression in response to H 2 O 2 occurred in an unusual bi-phasic pattern, with significant increases at 6 (96.9%±1.5, p<0.001) and 18 hours (234.0%±3.6, p<0.001), with a return to baseline in between. An increase in OPN mRNA preceded the increase in OPN protein at 18 hours, suggesting transcriptional regulation; however, the acute increase in OPN at 6 hours was not preceded by increased mRNA, suggesting multiple mechanisms of OPN regulation by H 2 O 2 . To determine if the increase in OPN at 6 hours is due to increased mRNA stability or translation, we performed an RNA stability assay. H 2 O 2 stimulation did not alter OPN stability or the rate of OPN RNA degradation, leading us to conclude the increase in OPN expression at 6 hours is due to increased translation. Further studies reveal H 2 O 2 -mediated increases in phosphorylation of 4E-BP1 at the redox-sensitive Ser65 site (89.4%±6.1, p<0.05), allowing for the subsequent release of eukaryotic initiation factor eIF4E and increased phosphorylation at Ser209 (139.2%±3.9, p<0.05), resulting in increased OPN translation. In conclusion, H 2 O 2 enhances OPN expression through acute increases in translation, while long-term increases in OPN occur through increased transcriptional regulation.