Pathophysiological Aspects of Vascular Remodeling in Cardiopulmonary Lesions: Influence of Inhibitor of DNA Binding/Differentiation-3 (ID3) & Estrogenic Endocrine Disruptors

Cardiopulmonary lesions, which manifest from various types of diseases such as pulmonary arterial hypertension, atherosclerosis, pulmonary arteriovenous malformations, lymphangioleiomyomatosis, and peripheral arterial disease, pose a public health problem. Vascular remodeling, which refers to alternations to the structure of the vessel is an important pathophysiological feature of these diseases. The Inhibitor of DNA-binding/Differentiation-3 (ID3), which is part of the ID family of transcriptional regulators, has been demonstrated to play an essential role in the vasculature and therefore may influence the alterations of these lesions. This review will highlight the existing understanding of how ID3 may contribute to cardiopulmonary lesion perturbations via involvement in vascular remodeling. Furthermore, based on the accumulative number of reports that suggest oxidative stress plays a critical role in the pathophysiology of vascular remodeling, we will also consider the impact of exposure to estrogenic endocrine disruptors (EEDs) such as polychlorinated biphenyls (PCBs) and bisphenol A (BPA) on ID3 & cardiopulmonary disease. Improved understanding of how ID3 pathways contributes to these molecular mechanisms in the lesion will likely provide useful knowledge in the mediation of vascular remodeling associated with ID3 & EED exposure, which may play an essential role in cardiopulmonary disease prevalence.


Introduction
Inhibitor of DNA Binding/Differentiation-3 (ID3) is a transcriptional regulator known to prevent stem cell differentiation and promote cell cycle progression. A member of the ID family of helix-loop-helix proteins programmed by an immediate-early gene responsive to oxidative stress and Previously, Felty and Das demonstrated that vascular endothelial cells exposed to E2 (17βestradiol) or estrogenic polychlorinated biphenyl 153 (PCB153) resulted in protein phosphorylation, endothelial neovascularization, and increased ID3 expression. Furthermore, PCB153 increased oxidative stress or ROS (reactive oxygen species) that facilitate ID3 expression. Estrogenic chemical exposure has been demonstrated to increase ROS, altering surrounding DNA essential for transcriptional stimulation of cell growth genes. ID3 protein-protein interactions occur via the HLH motif. During this, ID proteins dimerize and block the DNA binding activity of basic HLH transcription factors such as E proteins, which include: E12, E47, E2-2, and HEB, encoded by the TCF3, TCF4, and TCF12 gene [19]. ID3 protein-protein interactions can regulate transcription by Eproteins preventing subsequent binding and activation of target gene promoters. Furthermore, Eproteins suppress the expression of embryonic genes SOX2, OCT4, and NANOG leading to cell differentiation as demonstrated in Figure 1 [19]. Additionally, ID3 promotes cells to pass via cell cycle checkpoints by inhibiting the expression of cell cycle inhibitor gene p21Cip21. Research has demonstrated that ectopic overexpression of ID3 increased SOX2 and OCT4 expression and resulted in cell population positive for molecular stem cell markers CD133 + VEGFR3 + CD34. Based on these findings, ID3 maintains cells in a noncommittal or undifferentiated state by preventing the repression of pluripotency factors by TCF3 [20][21]. Since ID3 is a transcriptional regulator of genes involved in stemness and cell proliferation, it is plausible for EEDs to contribute to vascular remodeling mechanisms such as uncontrolled proliferation through ID3, thus contributing to the prevalence of cardiopulmonary lesions, in which vascular diseases manifest from.

Vessel Patterning & Perturbations
Cardiopulmonary lesions manifest from both proliferative and obliterative vascular diseases, which are categorized by the disruption of the normal patterning of vessels and their cellular components. Various pathways that lead to these end-stages are infrequently noticeable. Due to the assembly of normal vessels that may trace development, developmental biology, which combines the embryologic component of cell-to-cell interactions, cellular origins, & lineage with genetic studies, holds a possibility of demonstrating the role of various individual genes in pathogenesis [22]. The endothelium of the blood vessel plays a crucial role in considerations of cardiopulmonary diseases.
A vital foundation of current vascular biology is that the endothelial lining is a changeable interface [22]. Overall, the endothelium appears at best to function in this specific capability given its distinctive position between blood & tissue as well as its ability to generate biological effectors.
Certain aspects such as imbalances in the interaction and production of these different mediators appear to contribute and influence various vital functions of the endothelium. One particular aspect, the role as a non-thrombogenic vessel for not only blood but anti-inflammatory, antithrombotic, & growth inhibitory behaviors help to maintain the reliability of the vascular wall, in the face of numerous disease risk factors and stimuli that may lead to injury [22]. The vessel is comprised of three structural layers. The innermost layer, the tunica intima is lined with endothelial cells that attach and produce to the basal lamina with collagen type IV and laminin supported by an elastic lamina. These are anchored and connected by a collagen fiber network, elastic fibers, and fibrillin [23][24]. The middle layer, the tunica media is mainly composed of smooth muscle cells and elastin, which is arranged in a 3-D continuous network between collagen fibers and thin layers of proteogylycanrich extracellular matrix (ECM) [25]. The outermost layer, the tunica adventitia is a collagen-rich area comprised of myofibroblast cells. The high content of collagen fibrils (chiefly collagen types I and III) helps prevent vascular rupture at high pressures. The overall amount of collagen determines the tensile strength of the artery.

Inflammatory & Vascular Dysfunction
An essential inflammatory process categorizes numerous cardiopulmonary diseases. The pathogenesis of these cardiopulmonary lesions consists of a variable increase in numbers of both lymphocytes & macrophages and intimal smooth muscle cell proliferation. This response can be attributed to perturbations in the signal transduction events that regulate inflammation. Outcomes of this response consist of generation of reactive oxygen & nitrogen species, which can lead to cellular proliferation, antithrombotic properties that can be altered, and abnormal vascular plasticity. While procedures (invasive) are essential in some cases, lifestyle adjustments (physical activity & diet) & medical treatment result in benefits that may contribute to decrease in inflammatory process production [26]. Endothelial cells (EC) prevent adhesion of leukocytes. Nonetheless the perturbations of atherosclerosis can initiate various expressions of adhesion molecules on ECs causing leukocyte adhesion to the arterial wall. VCAM-1 (Vascular Cell Adhesion Protein-1), a protein that acts as a Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 18 July 2018 doi:10.20944/preprints201807.0334.v1 mediator and functions in leukocyte-endothelial cell sign transduction is a key part of this interaction.
Oxidized lipids has the ability to prompt gene expression by the pathway initiated by the NF-B (nuclear transcription factor B) such as TNF-and IL-1 [27]. Increased levels of cell adhesion molecules (CAM) are predicative of cardiac events and are an independent risk factor in men with coronary disease [4].
Chemokines are cytokines responsible for intermediating the maturation, differentiation, & migration of cells involved in inflammatory response. Chemokines can also promote ROS (reactive oxygen species) production and various cytokines via leukocyte penetration of the vessel wall. One particular chemokine MCP-1 (monocyte chemotactic protein-1) has the ability to regulate movement & infiltration of monocytes & macrophages into the inflammation site. In the presence of cardiovascular risk factors, it is overexpressed specifically in atherosclerotic lesions. Activation induces NF-B and AP-1, which leads to the release of IL-6 and proliferation of VSMC [28]. Proteins that demonstrate a multifaceted of signaling networks critical for distinctive regulation and adaptive inflammatory responses are known as cytokines. Through their influence on the development, growth, and activation of leukocytes, cytokines have the ability to modulate inflammatory responses.
A vital moderator in systemic inflammation is TNF-α, which activities include cell migration, activation of metalloproteinases (MMP), and production of interleukin CAM expression. TNF-α is detected in smooth muscle cells at all stages of the formation of athermanous plaques as well as endothelial cells [29]. The alteration from a vascular homeostasis inflammatory state is influenced by a difference between pro-inflammatory and anti-inflammatory events of interleukins. Some interleukins include: IL-1 which includes the stimulation of CAM, growth factors, tissue factors, chemokines, and various other cytokines. IL-6 is a multifunction cytokine with a central role in inflammation. Higher levels of IL-6 increase the risk of both mortality in patients with coronary heart disease and risk of myocardial infarction [30].

Vascular Remodeling, Extracellular Matrix (ECM), & Cell Adhesion
Arterial stiffness due to perturbations in the extracellular matrix is one of the mechanisms responsible for increased peripheral resistance in cardiopulmonary lesions. Evidence suggests arterial stiffness is an independent predictor of CVD events. The extracellular matrix (ECM), a collection of extracellular molecules secreted by cells, which provides structure and biochemical support to the surrounding cells deliver mechanical integrity to the vessel wall. Furthermore the ECM contains ligands that induce cell signaling to control migrations, proliferation, differentiation, and survival [31]. Cells respond to the ECM by remodeling their microenvironment that develops Aldosterone and angiotensin II (Ang-II) have previously been highlighted as an important regulator of cardiovascular homeostasis and pathogenesis of various CVD diseases. In order to evaluate its role, previous investigation of the expression of AT1R/AT2R at the vascular level was demonstrated [4]. Results showed the decreased expression of AT2R and increased expression of ATR1, which promotes vascular hypertrophy endothelial dysfunction, and growth.
Stiffness of small arteries as evaluated by the stress/strain connection was comparable in severely obese and lean subjects. Based on these lines of evidence, results show that severe obesity is associated with significant alterations in functional and structural features of small arteries, which may be responsible for the manifestation of elevated risk factors of CVD and increased incidence of coronary, renal, and cerebrovascular events conveyed in obesity. [36]. ChREBP. Furthermore, fructose caused FoxO1/3α transporting from the nucleus to cytosol and inhibited its binding to Aldo-B promoter region. Evidently this data suggests that fructose activates ChREBP and inactivates FoxO1/3α pathways to up-regulate both MG production and Aldo-B expression, leading to vascular remodeling [37].
Small artery pathophysiology is frequently invoked as a cause of obesity-related diastolic heart failure. However, evidence to support this hypothesis is insufficient, particularly in humans.
To address this, Khvandi et al studied human small artery structure and function in obesity and formation and that the expression peaks late when the proliferative index is low or diminishing and extensive apoptosis is observed, thus defining a innovated feedback loop in which an ID3 isoform is created that acts to limit SMC growth. Overall, this delivers the first indication that regulated intron preservation can modify a pathologic process in vivo [43]. ID3 has also been studied together with the lipoxygenase (12/15-LO), which is known to produce pro-inflammatory alterations in blood vessels that lead to the development of atherosclerosis [44]. 12  ID3 regulates the downstream mitogenic processing through depression of p21WAF1/Cip1, p53, & p27Kip1. These overall findings reveal a novel redox-sensitive pathway involved in growth control [47]. ID3 also plays a role in high fat diet stimulated visceral adipose VEGFA expression, microvascular blood volume, & depot expansion [48]. ID3 is essential to obesity due to its demonstration to stimulate angiogenesis that is considered an important factor of HFD (high-fat diet)-induced visceral adiposity [48]. mature-OECs. Results demonstrated that OECs from stroke patients present higher levels of proangiogenic factors at early stages, decreasing in mature OECs when they become more similar to mature microvascular endothelial cells [56]. Furthermore ID3 may be a predictive factor for stroke, which is sudden death to the brain cells due to inadequate blood flow. Expression levels of 10 candidate genes (ANTXR2, STK3, PDK4, CD163, MAL, GRAP, ID3, CTSZ, KIF1B, and PLXDC2) were data mined, compared between groups, and evaluated for their predicative ability at each time point in 23 ischemic stroke patients. Results demonstrated direct expression levels of the candidate genes were able to distinguish between stroke patients and controls with levels of sensitivity and specificity upwards of 90% across all three time points. Based on these lines of evidence, these findings confirm the diagnostic strength of the pattern of differential expression in an independent patient population, and further suggest that it is temporally stable over the first 24h of stroke [57]. ID3 expression also demonstrated a molecular stemness signature comprising of CD133 + VEGFR3 + CD34 + cells.

Estrogenic Endocrine Disruptors (EEDs) in cardiopulmonary disease
There is a growing concern that estrogenic endocrine disruptors may also contribute to the Results demonstrated that dietary intake of polychlorinated biphenyls, estimated using a food frequency questionnaire showed associated with a higher risk of developing hypertension during follow-up [60].
The effects of PCB126 on vascular inflammation has been linked to hepatic dysfunction utilizing a liver injury mouse model. Male C57Bl/6 mice were fed either an amino acid control diet (CD) or a methionine-choline deficient diet (MCD) in this 14-week study. Mice were exposed to PCB126 (0.5 mg/kg) and analyzed for inflammatory, calorimetric and metabolic parameters. bioavailable estrogen control (Belcher et al. 2015). Exposure-related changes in the rates of ventricular contraction, suggest toward increased parasympathetic activity, were detected in males. Decreased systolic blood pressure was observed in males exposed to BPA above 5 µg/kg·d and in females from the highest BPA exposure group. Based on these lines of evidence, results show significant sexspecific changes in gene expression in response to BPA that were consistent with the observed exposure-related phenotypic changes in the collagenous/non-collagenous extracellular matrix, cardiac remodeling, altered autonomic responses, and lipid metabolism [69]. BPA may also play a role in aortic and coronary atherosclerotic vascular remodeling. Over  formation was determined by RNA interference and showed ID3 siRNA to inhibit tube formation in estrogen exposed cells. Overall, estrogen-induced tube formation requires the presence of ID3 factors and estrogen increases ID3 phosphorylation via a redox-dependent process. [2]. Overexpressing ID3 cells presented significant estrogen-induced G2/M phase transition. Estrogen treatment increased phosphorylation of ID3 and total protein that was inhibited by tamoxifen; and

ID3 & Estrogenic Endocrine Disruptors in Vascular Remodeling
Pyk2 mediated estrogen-induced ID3 mRNA expression. These results suggest that Pyk2 signals ID3 expression and ID3 is crucial for estrogen-induced neovascularization in hCMEC/D3 cells [72]. Since cardiopulmonary diseases also affects the brain such as arteriovenous malformations, intracranial atherosclerosis, and vascular dementia, further research is warranted between ID3, EED exposure and disease outcomes. As demonstrated in Figure 1, exposure of EED may trigger ID3 to modulate mechanisms in vascular remodeling. Furthermore, these alterations may affect various cellular and non-cellular components such as cell growth, cell adhesion, cell migration, ECM alterations, and cell death as demonstrated in Figure 2.   This may lead to novel pathways for how the interaction of ID3, EEDs, and vascular remodeling mediates cardiopulmonary diseases and can further help to treat and prevent these detrimental diseases.

Conflicts of Interest
The authors declare that they have no conflicts of interest