Introduction: Tumor Necrosis Factor-a (TNF-a) plays an important role in cerebral aneurysm formation. Factors including inflammation and matrix degradation are considered to be important in aneurysm formation. Reactive Oxygen Species (ROS) play a pathophysiological role in vascular inflammation, proliferation, migration and angiogenesis. Among the major sources of ROS in vascular smooth muscle cells (VSMCs) are NAD(P)H oxidase (Nox). We investigated the role of TNF-a in regulation of Nox1 and Nox2 isoforms in cerebral VSMCs both in vitro and in vivo.
Methods: Rat cerebral VSMCs were treated with TNF-a at 10 and 50 ng/ml for 2 and 24hrs. These doses have previously shown phenotypic and inflammatory changes in cerebral VSMCs . RNA was extracted and qPCR was performed. As part of in vivo studies, Pluronic Gel (40% w/v) containing 30-60ug/ml of TNF-a was applied to adventitial surface of rat carotid arteries for 6 to 24 hours. Vessels were harvested for mRNA quantification.
Results: TNF-a decreased the expression of VSMC marker genes and myocardin, increased expression of KLF4 (a transcription factor) and proinflammatory/matrix remodeling marker genes including MMPs, VCAM1 and MCP1. In addition TNF-a increased the expression of both Nox1 and Nox2 at 2 and 24hrs in vitro but was more significant at early time points. At 24hr 10 ng/ml dose demonstrated increased the expression compared to 50 ng/ml. Similarly Nox1 & Nox2 expression increases early after stimulation with TNF-alpha
Conclusions: These findings indicate that cerebral VSMCs contain Nox1 and Nox2, are regulated by TNF-a and play an important role in NAD(P)H oxidase driven ROS production. TNF-a induces marked phenotypic modulation of vertebral VSMC while concomitantly increasing expression of Nox1 and Nox2. Alteration of Nox expression may abrogate aspects of TNF-a induced VSMC phenotypic modulation. These molecular changes implicate oxidative stress in vascular pathology underlying cerebral aneurysms and may provide a potential for future therapeutic strategies.
Patient Care: Understanding the underlying inflammatory mechanisms involved in aneurysm formation, progression and rupture will help us develop therapies to develop preventive measures and minimally invasive techniques.
Learning Objectives: 1) TNF-alpha induces oxidative stress by NAD(H) regulation
2) Nox1 and Nox2 may play an important role in smooth muscle cell phenotypic modulation. We have previously demonstrated phenotypic modulation by TNF-alpha
3) Smooth muscle cell phenotypic modulation has been shown to play an important role in aneurysm formation and progression
4) Use of anti-oxidants like SuperOxide Dismutase (unpublished data shown to reverse TNF-alpha stimulated phenotypic changes) may play a role prevention of aneurysm formation and growth