**Unique Phenotypes of Endothelial Cells in Developing Arteries: A Lesson from the Ductus Arteriosus Arteries: A Lesson from the Ductus Arteriosus**

**Unique Phenotypes of Endothelial Cells in Developing** 

Norika Mengchia Liu and Susumu Minamisawa Minamisawa

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Norika Mengchia Liu and Susumu

http://dx.doi.org/10.5772/66501

#### **Abstract**

[106] Kim J, Hwangbo +C, Hu X, Kang Y, Papangeli I, Mehrotra D, et al. Restoration of impaired endothelial myocyte enhancer factor 2 function rescues pulmonary arterial

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hypertension. Circulation. 2015;131(2):190–9.

174 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

2014;51(4):474–84.

Endothelial cells (ECs) play a critical role in regulating vascular pathophysiology. Various growth factors and relaxation factors such as vascular endothelial growth factor (VEGF) and nitric oxide (NO), which are derived from ECs, are known to maintain homeostasis and regulate vessel remodeling. Although the inner lumens of all types of vessels are covered by an EC monolayer, the characteristics of ECs differ in each tissue and developing stage of a vessel. Previously, we identified the heterogeneity of ECs of the ductus arteriosus (DA) by analyzing its gene profiles. The DA is a fetal artery that closes immediately after birth due to the changes in concentrations of oxygen and vasoactive factors such as NO and prostaglandin E. Studying the unique gene profile of ECs in the DA can therefore uncover the novel key genes involved in developing vascular function and morphology such as O2 sensitivity and physiological vascular remodeling. A comprehensive gene analysis identified a number of genes related to morphogenesis and development in the DA. In this chapter, we discuss the heterogeneity of vascular ECs in the developing vessel in the DA.

**Keywords:** vascular endothelial cells, ductus arteriosus, vascular remodeling, comprehensive gene analysis, oxygen, vitamin A

## **1. Introduction**

The endothelial cells (ECs) in vessels control the vascular tone, permeability, attraction of blood cells, which exhibit both innate and adaptive immunity, and migration/proliferation of underlying cells such as pericytes and smooth muscle cells (SMCs). To accomplish these roles, vascular ECs exhibit phenotypic heterogeneity during development in a timeand tissue-specific manner. The most significant diversity of ECs involves the differences

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

between arteries and veins as well as between large and small vessels. ECs undergo constant changes in phenotype depending on different situations, both physiological and pathological. Physiological angiogenesis occurs during development and repair processes. Many events in vascular development during gestation are reciprocated in the adult neovascularization that takes place in wound healing and ischemic disease treatment. In these cases, ECs must express pro-angiogenic factors. Pathological angiogenesis is often implicated as the abnormal proliferation of ECs such as that seen in tumorigenesis. Accordingly, many cancer studies have focused on vascular endothelial growth factor (VEGF), a pro-angiogenic factor produced from ECs. Endothelial damage and dysfunction causes cardiovascular diseases. For example, endothelial dysfunction reduces nitric oxide (NO) production, which decreases vasodilatory effects on SMCs. In addition, a decrease in NO production is also involved in the attraction of leucocytes and the production of various growth factors that leads to unregulated intimal thickening (**Figure 1**). Therefore, ECs play a central role in modifying the phenotypes of vessels. ECs have different roles depending on where they are located. For instance, in a developing vessel, ECs become tip cells or other stalk cells to regulate different molecular signaling to guide vessel sprouting [1]. Endothelial tip cells coordinate to have less proliferative activity by repressing Notch activity, thus upregulating VEGFR-2 (Flk-1) and other downstream

**Figure 1.** Summarized pathological and physiological vessel response. Damaged ECs are shown in dark blue. Due to the damage, there is a reduction of NO and an increase of ROS, which leads to platelet aggregation or leukocyte adhesion to the intima. Cytokines produced from platelets or leukocytes induce growth factor production and cause SMC hyperplasia and contraction. By contrast, healthy ECs constantly produce EDRF such as NO, so that SMC mitogen and contraction are absent. In developing vessels, ECs are proliferating or deriving from progenitor cells. Proliferating ECs can be distinguished into stalk cells and tip cells, which have different downstream VEGF pathways depending on Notch activity.

Notch transcription factors such as HASER1 [2]. By contrast, Notch signaling is more active and VEGFR-1 (Flt-1) expression is upregulated in stalk ECs. Although Notch and VEGF signals are greatly conserved in vessel sprouting among various tissues and species, how widespread it is in terms of tissue specificity remains to be elucidated (**Figure 1**). Increasing evidence shows that different signaling rules influence tissue-specific vessel sprouting—one study demonstrated that bone morphogenetic protein (BMP) signaling provides the cue for vein-specific angiogenesis during early development, and is independent from canonical VEGF-A signaling [3]. Casanello et al. reported that endothelial diversity is also present in the umbilico-placental vasculature, and emphasized that the heterogeneity of ECs is complicated and cannot be explained simply by comparing the differences between micro- and macro-vasculature, or artery versus vein [4]. Thus, EC shows great heterogeneity in health and disease, and studying the mechanisms of EC heterogeneity would contribute to the understanding of both vascular physiology and pathology.

We previously revealed the unique gene profile of ductus arteriosus (DA)-specific ECs. The DA, a fetal artery that connects the pulmonary artery (PA) and the aorta, is essential for fetuses to bypass the oxygenated blood delivered from the placenta directly to the descending aorta and not through the lung. The DA experiences a dramatic morphological change along with environmental factors after birth, though other connecting arteries remain unchanged. Therefore, even under similar physiological stresses underlying the DA and its connecting arteries, heterogeneity of ECs must exist. In this chapter, we focus on reviewing the unique identified gene profile of DA ECs, which should provide novel insights into heterogeneity in vascular ECs.

Moreover, investigating DA remodeling would potentially help the understanding of diseased vessels, just like other animal models in cardiovascular diseases. For instance, a wire injury model is used for studying pathology of endothelial injury/dysfunction [5]; low-density lipoprotein receptor-deficient mice [6] and apolipoprotein E-deficient mice [7] are commonly used as atherosclerosis models; calcium chloride [8], elastase [9], angiotensin II [10], or microRNA-21 [11] are infused to create an abdominal aortic aneurysms model. Developing a disease model occupies a great deal of scientific findings on pathophysiology, and so the existing models should always open to be refined. The DA can be an alternate model of an occluding vessel, an extracellular matrix (ECM)-enriched vessel, or an oxygen-sensitive vessel. Thus, studying DA ECs would be valuable for understanding an irregular angiogenic pathophysiology.

## **1.1. Embryonic vasculogenesis**

between arteries and veins as well as between large and small vessels. ECs undergo constant changes in phenotype depending on different situations, both physiological and pathological. Physiological angiogenesis occurs during development and repair processes. Many events in vascular development during gestation are reciprocated in the adult neovascularization that takes place in wound healing and ischemic disease treatment. In these cases, ECs must express pro-angiogenic factors. Pathological angiogenesis is often implicated as the abnormal proliferation of ECs such as that seen in tumorigenesis. Accordingly, many cancer studies have focused on vascular endothelial growth factor (VEGF), a pro-angiogenic factor produced from ECs. Endothelial damage and dysfunction causes cardiovascular diseases. For example, endothelial dysfunction reduces nitric oxide (NO) production, which decreases vasodilatory effects on SMCs. In addition, a decrease in NO production is also involved in the attraction of leucocytes and the production of various growth factors that leads to unregulated intimal thickening (**Figure 1**). Therefore, ECs play a central role in modifying the phenotypes of vessels. ECs have different roles depending on where they are located. For instance, in a developing vessel, ECs become tip cells or other stalk cells to regulate different molecular signaling to guide vessel sprouting [1]. Endothelial tip cells coordinate to have less proliferative activity by repressing Notch activity, thus upregulating VEGFR-2 (Flk-1) and other downstream

176 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**Figure 1.** Summarized pathological and physiological vessel response. Damaged ECs are shown in dark blue. Due to the damage, there is a reduction of NO and an increase of ROS, which leads to platelet aggregation or leukocyte adhesion to the intima. Cytokines produced from platelets or leukocytes induce growth factor production and cause SMC hyperplasia and contraction. By contrast, healthy ECs constantly produce EDRF such as NO, so that SMC mitogen and contraction are absent. In developing vessels, ECs are proliferating or deriving from progenitor cells. Proliferating ECs can be distinguished into stalk cells and tip cells, which have different downstream VEGF pathways depending on

Notch activity.

Vasculogenesis and angiogenesis are nomenclaturally similar as they both refer to the genesis of blood vessels [12]. Vasculogenesis is the de novo formation of blood vessels differentiated from mesodermal cells. Angiogenesis is the sprouting of blood vessels that occurs as a result of the proliferation of existing vascular ECs. Despite the difference in these two processes, vasculogenesis and angiogenesis are often compared to further understand their underlying molecular mechanisms. Indeed, a significant amount of knowledge on tumor angiogenesis was achieved by studying embryonic vasculogenesis [13]. Therefore, it is important to study developmental vascular biology and to understand vessel-specific heterogeneity. Moreover, determining the heterogenic diversity of ECs would help open up more options in clinical therapy, ultimately enabling individually designed therapeutic treatments.

The vascular network is the first functional system established in the embryo. A primitive vascular network is formed shortly after gastrulation by deriving endothelial progenitor cells from the mesoderm. This first process is called the formation of angioblasts. Angioblasts then differentiate into ECs by expressing various transcription factors and pan endothelial markers for tubular formation, which is called the primitive vascular plexus [13]. Some of the homeobox (Hox) transcription factors are known to be involved in this process. For instance, Hox A9 regulates the expressions of endothelial NO synthase (eNOS), VEGFreceptor 2 (VEGFR2), and vascular endothelial-cadherin (VE-cadherin), and is responsible for the tubulogenesis of mature ECs [14]. Hox B3 also plays a role in tubulogenesis [15]. Hox D3 induces the differentiation of ECs from angioblasts [16]. The primitive vascular plexus then undergoes complex remodeling accompanied by specification among arteries, veins, and capillaries to become the functional vascular system [13]. Sry-related HMG box (Soxs)-F subgroups Sox7, Sox17, and Sox18, along with vascular endothelial zinc finger-1 (Vezf-1), were found to be essential to the remodeling process [17, 18]. Thus, vasculogenesis in general consists of three steps: formation of angioblasts, formation of the primitive vascular plexus, and vascular remodeling. During these steps, the heterogeneity of vascular ECs is established.

#### **1.2. Physiology of the DA**

After the vascular system appears during embryonic development, the heart starts to function, and fetal circulation is established. Fetal circulation is different from adult circulation since the blood is oxygenated in the placenta instead of the lung. Prenatal lungs do not yet need to function so the DA bypasses the pulmonary artery and the descending aorta to send most blood to the body instead of the lungs. Patency of the DA is maintained due to the low oxygen level and high concentration of prostaglandin E2 (PGE2 ) in the blood circulated from the placenta, as well as the production of NO from ECs of the DA. Once the infant has been delivered and lung ventilation has begun, the DA must close properly to enable the transformation to adult circulation. Normal closure happens in two steps: functional closure and anatomical occlusion [19]. The first closure is triggered by an increase in pO2 and a drop in PGE2 , as well as a drop in blood pressure within the DA caused by the reduction in pulmonary vascular resistance. This functional closure causes the loss of blood flow which therefore induces hypoxia and extensive intimal thickening, followed by fibrosis. The hypoxia on the vessel wall further inhibits endogenous prostaglandin and NO production, which leads to an irreversible closure. Two to three weeks later, the sealed DA eventually becomes a fibrous band called the ligamentum arteriosum (**Figure 2**) [19]. Failed DA closure after birth is a condition called patent DA (PDA), and occurs frequently in premature infants. Medical or surgical treatment of PDA is required when the left-to-right blood shunt is significant.

**Figure 2. Representative pathways during DA remodeling**. In early gestation, the DA remained open due to the high concentration of PGE2 from placental circulation, and by producing EDHF (NO and CO in the figure). Low oxygen concentration induced ET-1 signaling and TGF-β expression in ECs, leading to functional closure. Postnatal DA is exposed to oxygenated blood that has reduced concentrations of PGE2 and NO. Due to reduced NO production, ROS are produced and monocytes are attracted to the intima. Monocyte-endothelial interaction induces cytokine and growth factor upregulation, thus promoting SMC growth. Extensive neointimal formation at a later stage causes ischemic hypoxia and ATP depletion, and eventual cell death.
