Vascular smooth muscle cells (VSMC) populate the medial layer of blood vessels and are able to modulate their phenotype in response to environmental cues. Defining the molecular pathways that influence the phenotypic state of VSMC is important for understanding cardiovascular development, vascular repair of injury and numerous vascular pathologies. Notch receptors are expressed throughout the lifecycle of VSMC and contribute to their differentiation and proliferation. Activation of Notch receptors by the ligand Jagged-1 (Jag-1) is believed to be a pro-differentiation signal for VSMC; however, the mechanisms downstream of Notch signaling influencing the phenotypic state of VSMC are not defined. I sought to identify novel gene targets of Jag-1/Notch signaling that promote VSMC differentiation, and to define potential non-overlapping functions of Notch receptors in regulating these genes. I observed decreased proliferation and increased contraction upon activation of Notch signaling by Jag-1 in human aortic VSMC. Using microRNA (miR) arrays, I identified miR-143 and miR-145 (miR-143/145) as enriched by Notch signaling and is required for Notch-induced VSMC differentiation in vitro.

Endothelial cell-dependent Notch signaling plays many critical roles in homeostasis and remodeling in the vasculature and serves an important role in the communication between endothelial cells and mural cells, including vascular smooth muscle cells (SMCs), pericytes, and fibroblasts. Our lab previously showed that endothelial cells govern SMC function and promote smooth muscle differentiation. Endothelial cells are able to induce smooth muscle cells into a mature contractile phenotype through cell contact and Notch signaling. Furthermore, Notch signaling has been shown to regulate SMC-specific gene and miR-143/145 expression to govern SMC differentiation. In this study, I demonstrated that endothelial cell-dependent Notch signaling enhances both contractile and synthetic phenotypes in smooth muscle cells, suggesting endothelial cells not only increase the contractile ability of smooth muscle cells, but might also signal to smooth muscle cells to build the basement membrane and form a functional blood vessel. My data indicate that the Notch2 receptor has a unique function in the regulation of SMC proliferation, and the Notch3 receptor has a distinct role in the regulation of synthetic phenotypes in smooth muscle cells.

The Notch signaling pathway has long been intricately linked with the development and function of the vasculature. In vascular smooth muscle cells (VSMCs), Notch signaling has a great influence on phenotype and is a strong promoter of differentiation and expression of contractile genes necessary to produce a functional vessel wall. However, the role of Notch signaling in VSMC proliferation and survival is less well defined, and some cases contradictory reports are given. Also, the contributions of each individual Notch receptor have not been clearly described. Thus, to better understand Notch signaling in VSMC phenotype, we investigated the specific roles of the predominant Notch receptors in VSMCs as they relate to differentiation, proliferation, and survival. We found that Notch3 promotes Platelet-Derived Growth Factor (PDGF)-induced proliferation in VSMCs, while Notch2 inhibits it. We also found that Notch3 was able to promote cell survival in response to apoptosis cues, while Notch2 had no discernible effect. Interestingly, we also found the expression of Notch2 and Notch3 were changed in response to proliferation and apoptosis inducers. Notch2 mRNA was significantly decreased after PDGF-BB treatment, a proliferation inducer, and Notch3 protein was degraded rapidly in response to induction of apoptosis. Additionally, we demonstrated that Notch3’s induction of cell survival genes required MEK/ERK signaling and Notch3 was capable of increasing levels of phosphorylated ERK. Altogether, these findings demonstrate that Notch2 and Notch3 have unique functions in regulating VSMC phenotype. In a mouse model devoid of Notch2 and Notch3 in smooth muscle cells, we were able to show that Notch2 and Notch3 are required for normal closure of the ductus arteriosus. Animals without Notch2 in VSMCs presented with patent ductus arteriosus with increasing incidence combined with the loss of Notch3. These mice died within one day of birth and also presented with aortic dilation. These phenotypes wee correlated with a significant loss of the smooth muscle contractile genes MYH11 and ACTA2, and it has been previously reported that defects in contractile differentiation can produce the PDA phenotype. This suggests that the Notch2 and Notch3 share an overlapping role in promoting VSMC differentiation. The Notch-regulated microRNA 145 has been strongly implicated in VSMC differentiation and also recently shown to regulate cardiovascular fibrosis. In order to better understand these connections to differentiation and fibrosis, we created and characterized a transgenic mouse for conditional expression of miR145, miR145TG. We were able to select a line that faithfully expresses the transgene and produces mature miR145 transcript. This model will allow for further investigation into miR145’s roles in promoting VSMC differentiation and mediating fibrosis.

In the last several years, the development of reagents that recognize smooth muscle-specific proteins has enabled researchers to identify smooth muscle cells (SMC) in tissue undergoing both differentiation and repair. These developments have led to increased research on SMC. The latest volume in the Biology of the Extracellular Matrix Series takes a current and all-encompassing look at this growing area of research. Devoted entirely to the subject of SMC, the book covers a diversity of topics-from SMC architecture and contractility to differentiation and gene expression in development. It also examines the proliferation and replication of SMC and its role in pharmacology and vascular disease. A must for cell, developmental, and molecular biologists, this book also will appeal to cardiologists, pathologists, and biomedical researchers interested in smooth muscle cells. Presents a molecular, genetic, and developmental perspective of the vas smooth muscle cell Overview sections highlight key points of chapters, including the clinical relevance of the research and expectations for future study Appeals to both the basic biologist and to the biomedical researcher of vascular disease

Atherosclerosis is characterized by the narrowing of the arterial lumen termed "stenosis", due to the expansion of arterial plaques. One of the major contributing factors to the formation of lesions and the neo-intima during post-angioplasty restenosis is the phenotypic change of medial vascular smooth muscle cells. This process switches them from a quiescent/contractile phenotype to a secretory, proliferative, migratory one. My work consisted of elucidating some of the molecular mechanisms implicated in this switch. We showed that the expression of an adenylyl cyclase (AC) isoform, AC8, is implicated in both the inflammatory and migratory properties acquired by trans-differentiated VSMCs. In human atherosclerotic arteries we showed that only intimal VSMCs strongly express AC8; very few AC8 positive VSMCs were detected in the medial layer, either in atherosclerotic or healthy arteries. In the rat balloon injury model of restenosis, we showed a transitory increase of AC8 expression. In vitro, we demonstrated that AC8 expression is regulated by the Notch pathway; inhibiting Notch amplified AC8 expression and decreased Notch target genes Hrt1 and Hrt3. In the same model of restenosis, the transitory up-regulation of AC8 expression coincided with Notch3 down-regulation. These set of experiments demonstrated that the Notch pathway decreases IL1[beta]-mediated AC8 up-regulation in trans-differentiated VSMCs and suggests that AC8 expression, besides being induced by the proinflammatory cytokine IL1[beta],also depends on the down-regulation of the Notch pathway occurring in an inflammatory context. As a whole, my studies attribute a new role for AC8 in pathological vascular remodeling.

This volume of the series Cardiac and Vascular Biology presents the most relevant aspects of vascular mechanobiology along with many more facets of this fascinating, timely and clinically highly relevant field. Mechanotransduction, mechanosensing, fluid shear stress, hameodynamics and cell fate, are just a few topics to name. All important aspects of vascular mechanobiology in health and disease are reviewed by some of the top experts in the field. This volume, together with a second title on cardiac mechanobiology featured in this series, will be of high relevance to scientists and clinical researchers in the area of vascular biology, cardiology and biomedical engineering.

​Vascular management and care has become a truly multidisciplinary enterprise as the number of specialists involved in the treatment of patients with vascular diseases has steadily increased. While in the past, treatments were delivered by individual specialists, in the twenty-first century a team approach is without doubt the most effective strategy. In order to promote professional excellence in this dynamic and rapidly evolving field, a shared knowledge base and interdisciplinary standards need to be established. Pan Vascular Medicine, 2nd edition has been designed to offer such an interdisciplinary platform, providing vascular specialists with state-of-the art descriptive and procedural knowledge. Basic science, diagnostics, and therapy are all comprehensively covered. In a series of succinct, clearly written chapters, renowned specialists introduce and comment on the current international guidelines and present up-to-date reviews of all aspects of vascular care.