Atherosclerosis is a chronic inflammatory disease that originates at regions of arteries exposed to disturbances in fluid flow and results in progressive plaque formation in those areas. Recent work on cellular responses to flow has identified potential mechanosensors and pathways that may influence disease progression. These results led us to hypothesize that the same mechanisms that mediate adaptive responses in the vasculature become maladaptive at sites of disturbed flow. Subsequent changes in gene expression and matrix remodeling help to entrain these inflammatory pathways. These events synergize with systemic risk factors such as hyperlipidemia, smoking, and diabetes, leading to disease progression.

Matrix metalloproteinases (MMPs) can degrade strength-giving collagens and other structural proteins of the arterial extracellular matrix. Overproduction of MMPs by monocyte/macrophages could therefore promote atherosclerotic plaque rupture and myocardial infarction. Freshly-recruited monocyte macrophages appear to use a prostaglandin (PG)-dependent pathway to coordinately upregulate a broad and potentially highly-destructive spectrum of MMPs. Differentiated macrophages rely on a series of distinct pathways to selectively upregulate groups of MMPs. Moreover, recent evidence suggests that different macrophage phenotypes express characteristically different spectra of MMPs and their inhibitors. New therapies may result from targeting matrix MMP overproduction.

Objective— Smooth muscle cell (SMC) differentiation is a dynamic process that must be tightly regulated for proper vascular development and to control the onset of vascular disease. Our laboratory previously reported that a specific focal adhesion kinase (FAK) inhibitor termed FRNK (FAK Related Non-Kinase) is selectively expressed in large arterioles when SMCs are transitioning from a synthetic to contractile phenotype and that FRNK inhibits FAK-dependent SMC proliferation and migration. Herein, we sought to determine whether FRNK expression modulates SMC phenotypes in vivo.

Methods and Results— We present evidence that FRNK^–/– mice exhibit attenuated SM marker gene expression during postnatal vessel growth and after vascular injury. We also show that FRNK expression is regulated by transforming growth factor (TGF)-β and that forced expression of FRNK in cultured cells induces serum- and TGF-β–stimulated SM marker gene expression, whereas FRNK deletion or expression of a constitutively activated FAK variant attenuated SM gene transcription.

Conclusions— These data highlight the possibility that extrinsic signals regulate the SMC gene profile, at least in part, by modulating the expression of FRNK and that tight regulation of FAK activity by FRNK is important for proper SMC differentiation during development and after vascular injury.

Objective— Angiogenesis is an integral part of many physiological processes but may also aggravate pathological conditions such as cancer. Development of effective angiogenesis inhibitors requires a thorough understanding of the molecular mechanisms regulating vessel formation. The aim of this project was to identify proteins that regulate tubular morphogenesis of endothelial cells.

Methods and Results— Phosphotyrosine-dependent affinity-purification and mass spectrometry showed tyrosine phosphorylation of ninein during tubular morphogenesis of endothelial cells. Ninein was recently identified as a centrosomal microtubule-anchoring protein. Our results show that ninein is localized in the cytoplasm in endothelial cells, and that it is highly expressed in the vasculature in normal and pathological human tissues. Using embryoid bodies as a model of vascular development, we found that ninein is abundantly expressed in the cytoplasm of endothelial cells during sprouting angiogenesis, in particular in the sprouting tip-cell. In accordance, siRNA-dependent silencing of ninein in endothelial cells inhibited tubular morphogenesis.

Conclusions— In this study, we show that ninein is expressed in developing vessels and in endothelial tip cells, and that ninein is critical for formation of the vascular tube. These data strongly implicate ninein as an important new regulator of angiogenesis.

Objective— Estradiol (E₂) is known to accelerate reendothelialization and thus prevent intimal thickening and in-stent restenosis after angioplasty. Transplantation experiments with ER^–/– mice have previously shown that E₂ acts through local and bone marrow cell compartments to enhance endothelial healing. However, the downstream mechanisms induced by E₂ to mediate endothelial repair are still poorly understood.

Methods and Results— We show here that after endovascular carotid artery injury, E₂-enhanced endothelial repair is lost in osteopontin-deficient mice (OPN^–/–). Transplantation of OPN^–/– bone marrow into wild-type lethally irradiated mice, and vice versa, suggested that osteopontin plays a crucial role in both the local and the bone marrow actions of E₂. In the vascular compartment, using transgenic mice expressing doxycyclin regulatable-osteopontin, we show that endothelial cell specific osteopontin overexpression mimics E₂-enhanced endothelial cell migration and proliferation in the regenerating endothelium. In the bone marrow cell compartment, we demonstrate that E₂ enhances bone marrow–derived mononuclear cell adhesion to regenerating endothelium in vivo, and that this effect is dependent on osteopontin.

Conclusions— We demonstrate here that E₂ acceleration of the endothelial repair requires osteopontin, both for bone marrow–derived cell recruitment and for endothelial cell migration and proliferation.