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An Insight on Multicentric Signaling of Angiotensin II in Cardiovascular system: A Recent Update

The multifaceted nature of the renin-angiotensin system (RAS) makes it versatile due to its involvement in pathogenesis of the cardiovascular disease. Angiotensin II (Ang II), a multifaceted member of RAS family is known to have various potential effects. The knowledge of this peptide has immensely ameliorated after meticulous research for decades. Several studies have evidenced angiotensin I receptor (AT1 R) to mediate the majority Ang II-regulated functions in the system. Functional crosstalk between AT1 R mediated signal transduction cascades and other signaling pathways has been recognized. The review will provide an up-to-date information and recent discoveries involved in Ang II receptor signal transduction and their functional significance in the cardiovascular system for potential translation in therapeutics. Moreover, the review also focuses on the role of stem cell-based therapies in the cardiovascular system.


INTRODUCTION
In the last decades, researchers have successfully unraveled key functions and mediators of the reninangiotensin system (RAS). Ubiquitously available RAS plays numerous physiological roles including regulation of blood pressure, fluid volume, vascular wall integrity, cell growth, cardiac output, and vascular tone in the body (Forrester et al., 2018). RAS is also involved in maintaining cardiovascular homeostasis, a network of intracellular signaling pathways, and various processes through endocrine, paracrine, and autocrine mechanisms (Bussard and Buss, 2018). Regardless of complexities associated with its movement from the local system occurring virtually in each organ to the hormonal system existing in circulation, the active end product is still Angiotensin II (Ang II) (Colafella and Danser, 2017).
Historically, in 1898, renin was discovered as a pressor compound within the extracts of the renal cortex of rabbits by Robert Tigerstedt. Their work was renewed in 1934, when Henry Goldblatt demonstrated induction of chronic hypertension by constriction of renal arteries in a dog with silver clamps. In continuation to this, Page and Helmer and Braun-Menéndez et al., discovered angiotensin as another compound from renal secretion bearing quick pressor response. These studies focused on the involvement of Ang II in physiological and pathophysiological functions. Besides, RAS inhibiting agents have shown promising benefits in the management of end-organ damage, ischemia, atherosclerosis, and cardiovascular-related disease (Nehme, 2019). A timeline of key historical findings associated with the study and discovery of Ang II associated with RAS is shown in Table 1 (Burton et al., 1985;Gibbons, 1998;Basso and Terragno, 2001;Andrea et al., 2006;Atlas, 2007;Skrbic and Igic, 2009;Benigni et al., 2010).
In view of traditional applications, investigators are making a consistent effort to explore the associated pharmacological effects of Ang II. Unfortunately, it is hoped that the next 100 years of research into RAS will uncover hitherto unimaginable therapeutic opportunities (Ferrario, 2006). The review will provide recent findings on Ang II receptor signal transduction and its functional significance in the cardiovascular system. In addition to this, the review also focuses on the applications of stem cell-based therapies in the cardiovascular system. The majority of pathophysiological conditions including hypertension and cardiac remodeling of Ang II are mediated by AT 1 R, which makes specific signaling pathways much clearer. In light of these facts the purpose of the present review is to provide newer insights in future research with an instinct that it will help emerging novel strategies to establish Ang II as a promising therapeutic candidate in translational research in the near future.

METHOD: EXCLUSION AND INCLUSION CRITERIA
The articles, written in English, published from 1985 to 2020, were exploited for gathering all relevant information of Ang II related articles from search databases namely, Science Direct, Medline/PubMed, Google Scholar, and other sources. Various databases were used to identify peer-reviewed papers dealing with the review theme of angiotensin-induced cardiovascular issues. A pilot review of literature assisted in identifying search terms that were used to categorize articles through a standardized and systematic process. The strings/words used for search purposes were as follows: "angiotensin", "induced", "receptor", "signaling", "disease", "mediators", "animal model", "biomarkers", "hypertrophic markers", "cardiac genes", "stem cells and others".

ANGIOTENSIN II RECEPTORS AND SIGNALING PATHWAYS
RAS involves different peptides with opposing biological effects. To sum up, the pro-inflammatory, pro-proliferative, and vasoconstrictive molecules are Ang II, AT1 R, and angiotensin-converting enzyme (ACE). Contrarily, AT2 R, ACE2, Ang (1-7), MrgD and MasR, exerts cardio-protective effects. In brief, angiotensinogen produced from the liver is converted into Ang I and Ang II via renin, esterase-2, cathepsin G, kallikrein, chymase, and angiotensin-converting enzyme. Ubiquitous actions of Ang II can be attributed to activation of several signal transduction pathways modulated by receptors including AT 1 R and AT 2 R to initiate RAS or further get cleaved into peptides namely, Ang IV, Ang (1-7), and alamandine, which express their effects via AT4 R, Mas R and MrgD, respectively (Adamcova et al., 2021;Matsubara, 1998). Interestingly, administration of Ang (1-7) was evidenced to provide a protective effect during chronic infusion of Ang II in rats (Grobe et al., 2007). However, the pharmacology of AT 3 R and AT 4 R has not been categorized fully and hence they are not definitively classified under mammalian Ang receptors (Figure 1) (Touyz and Berry, 2002). Based on several investigations, AT 1 R

Discoverer (Year) Development in RAS
Richard Bright (1836) Related hypertrophy to an increased resistance to blood flow in the small vessels due to the altered condition of the blood George Johnson (1868) The pathology behind left ventricular hypertrophy F. A. Mahomed (1872) ➢ Described high blood pressure using a primitive sphygmograph ➢ Linked left ventricular hypertrophy to hypertension due to nephritis ➢ Presence of high blood pressure in patients without renal disease Riva Rocci (1896) Introduced first indirect sphygmomanometer to measure arterial pressure in humans Tigerstedt and his assistant Bergman (1898) ➢ Analyzed and discovered the presence of a pressor compound in the renal tissue 'Renin' ➢ Explained association between renal disease and cardiac hypertrophy Korotkoff (1905) Defined the cardiac sounds Goldblatt et al. (1934) ➢ Linked ischemic characteristic of renal disease with hypertension ➢ Induced experimental hypertension in a dog by partial constriction of a renal artery using a silver clip ➢ Proposed the existence of a humoral mechanism Irvine. H. Page heading Indianapolis group (1940) Discovered renin as an inactive enzyme, activated by plasma protein compound renin activator and they named angiotensin Edward Braun Menendez heading Argentine group (1940) and AT 2 R have been evidenced to be associated with the majority of Ang II mediated signaling pathways (Kawai et al., 2017). AT 1 R is clearly different from AT 2 R in signalling mechanisms, tissuespecific expressions, and molecular weight. AT 2 R may also counter-regulate functions mediated via AT1 R. However, the signaling mechanisms of AT 2 R are still speculative compared with those of AT 1 R. Moreover, most of the classic cardiovascular effects of Ang II are conveyed by AT 1 R, including, vasoconstriction, hyperplasia, sodium retention, vascular cell hypertrophy, myocardial fibrosis, arterial wall thickening, aggravation of inflammatory responses, and stimulation of ROS.
AT 1 R is a G protein-coupled receptor and is widely expressed in the heart, endothelium, smooth muscle, kidney (mainly in glomerulosa cells), brain, adipose tissue and adrenal glands (Li et al., 2012). AT 1 R encourages intracellular pathways via activation of subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, several protein kinases, transactivation of growth factor receptor, or direct interaction with AT 1 R interacting proteins like Guanine nucleotide exchange factor (GEF)-like protein (GLP), AT 1 R associated protein (AT 1 R AP), phospholipase C (PLC γ1) and Janus activated kinase (JAK 2) ( Figure 2). In addition, it also turns on several downstream signals, like mitogen-activated protein kinase/extracellular signalregulated kinases (MAPK/ERK), Ras/Rho, and translocation of MAPK in the nucleus (Ahmadian et al., 2015). AT 1 R of mouse and rat exists as Ang II type I subtype A receptor (AT 1A R) and Ang II type I subtype B receptor (AT 1B R), which have similar activation, ligand binding properties, and identical amino acid sequences, but differ in tissue transcriptional and distributive regulation. AT 1A R is widely expressed and regulate blood pressure. Thus, it is anticipated to be the closest homolog to the human AT 1 R (Touyz and Berry, 2002).
G-Protein, Protein Kinases, Nicotinamide Adenine Dinucleotide Phosphate , and Growth Factor-Mediated Signaling Ang II activation of AT 1 R promotes variously convoluted, convergent, and diverse signaling pathways. However, research has established specific components essential for Ang II dependent signaling pathways. In brief, AT 1 R binds with heteromeric G-protein (G q/11 , G i , G 12, and G 13 ) and allows activation of secondary messengers such as Rho GEFs, PLCβ, inositol 1,4,5-trisphosphate (IP 3 ), diacyl glycerol (DAG), and reactive oxygen species (ROS). This further regulates downstream effectors like phospholipases. The response may differ depending on effector tissue, such as in vascular smooth muscle cells (VSMCs) contraction is regulated through G 12/13 Rho/Rho kinase-mediated myosin light chain phosphatase (MLCP) inhibition or G q/11 Ca 2+ sensitive myosin light chain kinase (MLCK) activation. Similarly, Src family kinase also regulates vascular contraction through MLCP inhibition and Rho kinase/ RhoA signaling. Ang II mediated AT 1 R potentiates various serine/threonine kinases such as PKC, Akt, and mitogenactivated protein kinase (MAPK) family kinases and other intracellular protein kinases like, non-receptor and receptor tyrosine kinases ( Figure 2 and Table 2).
Ang II stimulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases to produce ROS causing renal deterioration, cardiac hypertrophy, and VSMC migration. In general, Ang II increases the production of ROS via activation of the catalytic subunit of NADPH, the Nox family proteins. The catalytic subunits of NADPH include dual oxidase (Duox1 and Duox2) and the Nox family (Nox1-Nox5). Subsequent stimulation of Nox family proteins increases its interactions with associated specific regulatory subunits p67 phox, p47 phox, p22 phox, and Nox1 ( Figure 2) (Kawai et al., 2017). Ang II stimulated Nox4 generation in vascular cells and renal tissues via AT 1 R is a source of oxidative stress, hypertension and organ failure. Ang II upregulates JNK and Nox4 in BubR1 (budding uninhibited by benzimidazole-related 1) lowexpression mice (Aoyagi et al., 2019). Moreover, it stimulates the expression of AT 1 R and receptors for advanced glycation end products (RAGE) by stimulating the PKC-ERK-NF-κB signaling pathway. In addition, it can increase intracellular ROS and critical mediators of cardiomyocyte hypertrophy by regulating expression levels of NADPH oxidase 2/4 in H9c2 cells . Ang II mediated transactivation of nonreceptor tyrosine kinase (Janus kinase, c-Src, and focal adhesion kinase) and tyrosine kinase (EGF, PDGF, IR, etc.) resulted in modulation of various downstream targets like MAPK (Vukelic and Griendling, 2014). It can act as a growth factor that can regulate cell hypertrophy in VSMCs. Investigations have established dynamic phenomenon for AT 1 R dependent transactivation of growth factor receptor-like EGF receptor (EGFR), followed by subsequent activation of Akt/p70S6, mechanistic target of rapamycin (mTOR), and Ras/ERK signaling resulting in cardiac hypertrophy and fibrosis (Eguchi et al., 1999;Eguchi et al., 2001;Ohtsu et al., 2006). Ang II stimulates selective mTOR2-dependent phosphorylation of SGK1 but not Akt (Gleason et al., 2019). The transactivation is mediated by secondary messengers like ROS, PKC, Src kinase, and metalloproteinase-dependent release of EGFR ligands such heparin-binding EGF, TGF-α, and EGF. Peng et al., 2016 evidenced that c-Src dependent EGFR transactivation in ERK/ Akt pathway may a play crucial role in Ang II induced cardiac remodeling in H9c2 cells (Peng et al., 2016). AT 1 R mediated A Disintegrin And Metalloproteinase 17 (ADAM17) dependent EGFR activation results in VSMC migration and hypertrophy via PI3K/Akt/mTOR/p70S6K pathway and Ras/ERK pathway. Ang II mediated ADAM17 requires ROS and p38 MAPK phosphorylation. Zhang Y. et al. (2019) demonstrated that Ang II promotes Mer tyrosine kinase shedding via AT 1 R/ROS/ p38 MAPK/ADAM17 pathway in macrophages of ApoE −/− mice . Furthermore, BMX (bone marrow kinase), a non-receptor tyrosine kinase has been identified as an upstream signalling molecule for Ang II-mediated EGFR activation. Thus, systemic inhibition of EGFR or ADAM17 decreases Ang IIinduced cell migration and aortic aneurysm.
Ang II-dependent connective tissue growth factors (CTGF) and transforming growth factor-β (TGF-β) are initial pro-fibrotic mediators involved in cardiac fibrosis (Wong et al., 2018;van Beusekom and Zimmering, 2019). The expression of CTGF and TGF-β are interlinked. Ang II increases mRNA expression of TGF-β and NF-κβ, an important mediator of the hypertrophic growth of the heart, in H9c2 cells (Prathapan et al., 2013). Further, myocardial CTGF expression after Ang II exposure is likely dependent on latent activation of TGF-β via canonical Smad-pathway in NIH/3T3 fibroblasts (Wong et al., 2018). Overexpression of fibroblast growth factor 23 (FGF23) augmented cardiac fibrosis and hypertrophy in Ang II administered mice via PPARα/PLCγ-NFAT1/TGF-β signaling . Contrarily, FGF21 enhances cardiac function and reduces Ang II induced cardiac hypertrophy through in silent information regulator 1 (SIRT1)/ adenosine monophosphate-activated protein kinase (AMPK) pathway . In addition, Ang II is well known to transactivate insulin-like growth factor I (IGF-I) receptor (IGF-IR) and platelet-derived growth factor (PDGF) receptor (PDGFR) in VSMC (Du et al., 1996). However, unlike EGFR, research on the role of Ang II-mediated IGF-IR, TGF, and PDGFR in cardiovascular pathophysiology is still limited.
Ang II-induced signaling via AT 1 R is correlated with MAPK activation and enhanced phosphorylation of protein tyrosine. This fact highlights that besides vasoconstriction, Ang II also possesses the inflammatory and mitogenic properties. Similar to AT 1 R, the existence of AT 2 R is also opting for increased attraction due to its opposite effect than the former. AT 2 R also belongs to the GPCR family and stimulates the SH2 domain-containing phosphatase (SHP-1) and MAPK phophatase1 (MKP-1) resulting in attenuation of tyrosine phosphorylation. In addition, AT 2 R accelerates vasorelaxation through PKA-dependent eNOS   activation and paracrine signaling through bradykinin/cGMP/NO production. Considering AT 2 R, some AT 2 R interacting proteins have shown physiological roles in the suppression of tumors, inflammation, ROS production and hypertrophy.

G-Protein Independent Signaling via β-arrestin
Ang II stimulated AT 1 R can activate various signaling cascades such as G-protein independent and G-protein dependent signaling. Unlike G-protein dependent signaling, the G-protein independent signal transduction cascade includes G-protein and β-arrestin (Forrester et al., 2018). Isoforms of β-arrestin i. e., β-arrestin 1 and 2 are recruited to AT 1 R and stabilize them with high-affinity conformations (Sanni et al., 2010). Mechanistically, β-arrestin were described as a protein that uncouples GPCR from G-protein for mediating receptor internalization and G-protein independent signaling. β-arrestin mediated signaling includes activation of p38 and Akt/MAPK, JUNK, ERK1/2, and Src tyrosine kinases. A study involving human embryonic kidney (HEK)-293 cells biased agonist Sar 1 , Ire 4 , Ile 8 -ANG II (SII) or a mutant AT 1 R-DRY/AAY suggested various active conformation of AT 1 R. The SII or mutant AT 1 R induced G protein independent, but β-arrestin 2-dependent ERK activation (Wei et al., 2003). AT 1 R, both β-arrestin and G q/11 proteins mediate biased signaling (Ferraino et al., 2021). β-arrestin-biased AT 1 R signaling promotes vascular remodeling with the activation of MAPK and Src-based signaling. Interestingly, mechanical activation of AT 1 R caused increased affinity toward β-arrestin biased ligand TRV 120023, suggesting stabilization of a biased active receptor conformation (Ma et al., 2021). Apart from its involvement in β-arrestin-dependent signaling, AT 1 R was also reported to be involved in stretch-induced pathways in different cells. Interestingly, GRK2 and PKC, the kinase responsible for β-arrestin binding of many GPCRs have also been found to be activated up on a stretch in rat ventricular myocytes (Turu et al., 2019). In a study, the vasoconstrictor responses were increased by G q/11 AT 1 R biased agonists TRV120055 and TRV20056. Here, G q/11 AT 1 R was an essential component of dynamic mechanochemical signaling in VSMC causing myogenic tone (Cui et al., 2020). Alongwith AT 1 R, AT 2 R is also suggested to be primarily stimulated via G-protein independent signal transduction cascade including β-arrestin and GPCR kinase.

ANGIOTENSIN II IN CARDIOVASCULAR SYSTEM
As a vital bioactive peptide of RAS, Ang II is associated with diverse mechanistic insights into understanding how Ang II contributes to multiple cardiovascular physiology and pathophysiology functions. Century-old research on RAS has uncovered Ang II and its involvement in the pathophysiology of cardiovascular diseases. Ang II is involved in the regulation of cell communication, impulse propagation, cardiac contractility, apoptosis, growth, and remodeling (Kawai et al., 2017). Summary of in vivo and in vitro pharmacological investigations are presented in Figure 2 and Table 3. In most of the in-vivo studies, the approach used for induction was a subcutaneous infusion of Ang II.

Angiotensin II in Hypertension
Hypertension is one of the most critical predisposing factors for the development of cardiovascular disease. Several factors contributing to the pathogenesis of hypertension include salt intake, stress, and Ang II (Kulkarni et al., 1998;Meneton et al., 2005). Ang II as a powerful vasoconstricting agent can induce aldosterone secretion and thereby retention of salt and water, which regulates blood pressure. In addition to this, Ang II has also been shown to have important oxidative, inflammatory, and immune-mediated actions. Ang II even at doses that do not alter blood pressure, allows the migration of inflammatory cells and induces the expression of inflammatory markers in the aorta of normotensive mice (Lima et al., 2019) Nair et al. in 2015 found toll-like receptor 4 (TLR4), highmobility group box 1 (HMGB1), and proinflammatory cytokines mediated immune response contributed to Ang II-induced hypertension in rat tubular epithelial cell line NRK52E . In support of this concept, they designed another study to determine the involvement of HMGB1 signaling in Ang II-induced hypertension in the para ventricular nucleus (PVN). The interaction between the inflammatory cytokine protein, HMGB1, and TLR4, resulted in the up-regulation of NF-kB which in turn resulted in hypertension in Neuro-2a cells of mice treated with Ang II (Nair and Philips, 2015). These findings were consistent with reports by Li et al. (2016) and Yu et al., 2015 that showed Ang II increases hypertension and hypothalamic infiltration via TLR4/MyD88/NF-κB signaling pathway and peroxisome proliferator-activated receptor-γ (PPAR-γ) in the PVN in hypertensive rats (Yu et al., 2015;Li et al., 2016).
In addition to, TLR4 and PPAR-γ, Ang II is known to induce ROS via activation of NADPH oxidase mediated Nox-1 and p22phox in Ang II-induced DNA damage in-vivo and in-vitro models (Wattanapitayakul et al., 2000;Sarr et al., 2006;Zimnol et al., 2020). Hyperactivation of Nox acts as a major source of ROS production in cardiac tissue, promotes apoptosis and increases oxidative stress via the MAPK pathway (Wen et al., 2019). Ogola et al., 2019 evidenced that pretreatment with G protein-coupled estrogen receptor (GPER) agonist G1 inhibits Ang II-induced ROS, NADPH, Nox4 mRNA expression via cAMP and phosphodiesterase inhibition (Ogola et al., 2019). Moreover, Ang II also induces pressor response and vasoconstriction by reduction of an active form of Ras-related C3 botulinum toxin substrate 1 (Rac1) and Nrf2 nuclear translocation (Pepe et al., 2019). Rac, a small G protein is an essential molecule for the function of NADPH oxidase components along with, phosphorylated Smad 2/3, atrial TGF-β1, and atrial superoxide in Ang II hypertensive rats (Yagi et al., 2010).
Another enzyme source of ROS includes uncoupled nitric oxide synthase (NOS), endoplasmic reticulum oxidase, xanthine oxidase, and mitochondrial oxidase. ROS affects the function of a cell by modifying proteins through post-translation modifications such as phosphorylation and oxidation (carbamylation, glutathionylation, nitrosylation, and sulfenylation). Proteins that are affected include matric metalloproteinases, cytoskeletal structural protein, transcriptional factors, signaling molecules, and ion transporter receptors (Griendling et al., 2016). ROS activate all 3 members of the MAPK family, including JNK, p38MAPK, and ERK1/2, essential for regulating vascular and cardiac cells (Jennings et al., 2010). In a study, catechins were reported to inhibit Ang II-induced VSMC proliferation by inhibiting Ang II activated MAPK and activator protein-1 signaling pathways (Won et al., 2006) (Figure 2). MEK-ERK are phosphorylated in arteries of hypertensive individuals and in a mouse model. Activation of this pathway results in the promotion of human arterial SMC (HASMCs). A known cysteine protease, Cathepsin L/V is interdependent on extracellular matrix accumulation and tissue inflammatory responses, allowing regulation of arterial remodeling. Lu et al., 2020 showed that Z-FF-FMK, a cathepsin inhibitor significantly reduces MEK-ERK phosphorylation (Lu et al., 2020).
Also, Ang II is responsible for causing atherosclerosis through VCAM 1 activation via protease-dependent NF-κB-like transcriptional mechanisms (Tummala et al., 1999). Acute doses of Ang II act primarily on VSMC to reduce blood pressure whereas chronic infusion of Ang II is neutrally mediated (∼10 h) (Li et al., 1996). It has been reported that statins reduce the incidence of cardiovascular remodeling. It is well known that apart from cholesterol-lowering, it also provides pleiotropic effects on the cardiovascular system, including antioxidant, anti-inflammatory, and improvement of endothelial function (Yagi et al., 2010). Candesartan, an AT1 R blocker and apocynin, NADPH oxidase inhibitor evidenced reduced pressor effect by AT1R-dependent ROS-SAPK/JNK, ERK1/2, and p38MAPK signaling . Likewise, Pitavastatin exerts eNOS based protective action in Ang IIinduced cardiovascular remodeling through suppression of (TGF)-β1-Smad 2/3 signaling pathway and oxidative stress (Yagi et al., 2010). Ang II inhibition extensively improved hypertension, hyperfiltration and control renal damage. Inhibition of Ang II enhanced the NF-KB activity which may additionally result in inhibition of its downstream gene expression, particularly NADPH-oxidase.
p38 and MAPK pathways also play a crucial role in extracellular matrix deposition and cell proliferation during hypertensive cardiovascular remodeling (Jing et al., 2011;Jiajing et al., 2020). ERK represents MAPK and is involved in the growth and proliferation of VSMC. However, p38 MAPK inhibitor, PI3K inhibitor, and the c-JNK inhibitor didnot affect arterial blood pressure or renal sympathetic nerve activity in heart failure (Shinohara et al., 2015) (Figure 2). LCZ696, an AT1 R blocker, attenuated the hypertrophic expression of ANP, βMHC, and TIMP2 in Ang II-induced remodeling in cardiomyocyte and collagen I, collagen III, and TGF-β in cardiac fibroblasts via ERK inhibition . A potent angiogenesis inhibitor, endostatin reduced the levels of ANP and BNP in primary neonatal rat cardiomyocytes and Ang II-treated rats via the cAMP/PKA pathway (Dai et al., 2019). Similar results were observed by ghrelin, a gut peptide that decreased Ang IIinduced cardiac fibrosis by inhibiting TGF-β1 and upregulating PPAR-γ in rats. Also, it inhibits smooth muscle cell proliferation stimulated by angiotensin II by preventing cAMP/PKA signaling .
Astonishingly, a recent study suggested that PUO4F2/Brn-3b transcription factor acts as a novel regulator of adaptive hypertrophic responses in the adult heart along with other markers such as, ANP/βMHC. Levels of Brn-3b mRNA and related proteins are increased in Ang II treated mouse hearts and foetal heart-derived H9c2 cells or primary cultures of neonatal rat ventricular myocytes with varied hypertrophic alterations. Alternate signaling pathways involved in activation of Brn-3b promoter include p42/p44 MAPK/ERK1/2 or calcineurin pathways (Mele et al., 2019;Cheng et al., 2020). Cardiac fibrosis is induced by the pro (renin) receptor, which could be more worsened by the involvement PRR-ERK1/2-NOX4 pathway and ROS during the development of alcoholic cardiomyopathy (Cao et al., 2019). Ang II-induced collagen accumulation in cardiac fibroblast stimulates oxidative stress, ROS and inflammation via NF-κB/MAPK and nuclear factor erythroid 2-related factor (Nrf2)/AMPK pathway (Su et al., 2018;Du et al., 2019). Fibrotic proteins including, matrix metalloproteinases, collagen, and TGF are overexpressed in presence of Ang II through Akt/PI3K and NF-κB/MAPK pathway .
Despite the growing interest in big data approaches, with the aim of studying the genetics of cardiovascular disease, fishes are becoming an increasingly popular choice to study associated genetic alterations. To clarify the putative actions of Ang II in cardiac remodeling, Anguilla Anguilla was selected. Immunoblotting and immunolocalization results suggested that Ang II downregulates both localization and expression of molecules affecting apoptosis and cell growth such as eNOS, heat shock protein-90, and c-kit (Imbrogno et al., 2010;Imbrogno et al., 2013;Filice et al., 2017). Another recent study demonstrated the effect of Ang II on morpho-functional remodeling in heart of Danio rerio. The findings were paralleled by the upregulation of AT 1 R and AT 2 R expressions. Moreover, a significant change in expression of cytochrome b-245β polypeptide protein, superoxide dismutase 1 soluble mRNAs, NFk-light polypeptide gene enhancer in B cell, and GATA binding protein, indicated cardiac remodeling (Filice et al., 2021).

Angiotensin II in Stem Cell Therapy in Cardiovascular System
Recent investigations have endeavored to improve stem cell functionality, provide stem cells as a promising therapeutic candidate for tissue transplantations in the cardiovascular system. Evolving research has evidenced the influence of RAS on stem cell growth, function, and proliferation (Becher et al., 2011). Several studies have demonstrated the role of Ang II in the differentiation of progenitor cell/stem cell (Matsushita et al., Frontiers in Pharmacology | www.frontiersin.org August 2021 | Volume 12 | Article 734917 2006). The presence of AT 1 R in differentiated, but not in undifferentiated cells, suggests the concept that Ang II can regulate differentiation of stem cells (Huang et al., 2007). In addition to influencing a different kind of stem cells, the RAS effect on cardiovascular-related stem cell transplantation has largely been evaluated regarding the intracellular pathways of Ang II. Mesenchymal stem cells (MSCs) are of great significance, along with their various autocrine-paracrine effects on the cardiovascular system and immune system . Local RAS component Ang II has been reported to be expressed in rat MSCs. Vascular endothelial growth factor (VEGF) has been recognized in an invasion of extracellular matrix, migration, proliferation and survival of MSCs. Pretreatment with Ang II increases mRNA expression of VEGF in MSCs through Akt/ERK1/2 signaling pathway via AT 1 R. Considering this, pre-treatment of MSCs with LY292002, an Akt inhibitor attenuates Ang II-induced expression of VEGF. Notably, the involvement of Ang II increases the expression/ production of VEGF in MSC grafts and improves transplantation efficiency (Liu et al., 2014). Thus, an angiogenic function of Ang II stimulates cells in ischemic regions through VEGF-induced endothelial nitric oxide synthase (eNOS). Ang II induces cardiomyogenic differentiation of rat bone marrow MSCs more efficiently than TGF-β1. The autocrine TGF-β/Smad pathway makes the differentiation of adipose tissue-derived MSCs to SMCs. Also, it acts synergistically with VEGF to ameliorate the differentiation of bone marrow-derived MSCs into endothelial cells (Ikhapoh et al., 2015).
Cardiac hypertrophy is a phenotypic response of the heart associated with various disorders. The genetic factor is an important determinant of phenotypic expression in hypertrophy (Marian, 2008). Neuron-derived orphan receptor-1 (NOR-1) transgenesis upregulates key genes involved in cardiac hypertrophy (Myh7, encoding for β-myosin heavy chain (β-MHC)) and fibrosis (Loxl2, encoding for the ECM modifying enzyme, Loxl2) in Ang II-induced cardiomyocytes (Cañes et al., 2020). In another study, Ang II and TGF-β1 upregulated the expression of the structured cardiomyocytes genes such as DES, TNNT2 and MYH6 as well as main cardiac genes-markers like GATA4, TBX5, and NKX2-5. Also, an increased expression of cardiac ion channel genes is evidenced with Ang II and TGF-β1 in human amniotic fluid-derived MSCs (AF-MSCs). Ang II and TGF-β1 treated AF-MSCs showed an increase in connexin43 protein and Nkx2.5 protein in AF-MSCs (Gasiūnienė et al., 2019). MSC can be an effective route for refining cell-based therapy of angiogenesis, vascular stabilization, and endothelial cell survival . Besides, TGF-β secretion is associated with the MAPK/ERK pathway; and Ang II in this pathway interferes with TGF-β production.
Bone marrow is one of the major Ang II producing organs and participant in the regulation of immunity and hematopoiesis (Yamashita et al., 2020). Being an inducer of differentiation of MSC, Ang II at doses ranged 0.1 to 10 μM can regulate apoptosis. Ang II could increase mitochondrial ROS through the activation of Nox2. In a study, Ang II at the dose of 1 and 10 μM leads to apoptosis in bone marrow-derived MSCs due to mitochondrial ROS production and mitochondrial DNA leakage mediated via AT 1 R. Treatment with AT 1 R inhibitor, losartan, markedly inhibited the Ang II-induced apoptosis and mitochondrial ROS . Ang II production by cleaving enzyme chymase is several times higher in bone marrow than in other tissues. In a study, flow cytometry results showed Ang II generated via a chymase-dependent pathway in bone marrow was 280 -fold higher than in the heart. CD68 + myeloid progenitor possesses higher chymase expression than CD68 − progenitor cell in bone marrow (Yamashita et al., 2020). Another study showed that AT 1a R was widely expressed by human bone marrow CD34 + , CD38 − cells, and lymphocytes (Rodgers et al., 2000). Ang II, but not Ang (1-7), increased adhesion of MNCs or CD34 + cells to fibronectin via ACE2/Ang-(1-7)/Mas pathway (Singh et al., 2015). The reported pathway stimulates vasoprotective functions of CD34 + cells.
Autologous bone marrow MSCs are effective for regression of aneurysms in Ang II-induced ApoE −/− mice (Akita et al., 2019). Bone marrow MSCs derived conditioned medium could prevent aneurysm growth through macrophage polarization regulation . In addition, precursor fibronectin type III domain-containing protein 5 or irisin, a novel myokine has the potential to improve bone marrow MSC mediated paracrine effect and engraftments in infarcted hearts (Deng et al., 2020). Previous studies have shown that MSC-derived exosomes have distinct properties, including immunomodulation, angiogenesis, and paracrine effect that protect organ functions in animal studies. Additionally, recent investigations have evidenced that adipose-derived MSCs (ADMSCs) derived exosomes also possess a capacity of immunomodulatory, cardioprotective, and antiinflammatory effects. Interestingly, irbesartan, an AT1 R blocker, was shown to abolish the effects of ADMSC-derived cell sheets in a rat model (Yamamoto et al., 2018). Administration of miR-19a/ 19b (exo/miR-19a/19b)  cardiac HL-1 cells significantly suppressed the apoptosis and fibrosis in infarcted hearts . Human-induced pluripotent stem cells (iPSCs) possess unique features to differentiate and self-renew into different types of cells in the body (Johansson et al., 2020). They are artificially derived from adult differentiated non-pluripotent somatic cells. In particular, these human cells-based models are anticipated to become an alternative for animal models. iPSCs can express Ang II receptors. As evidenced, Ang II induces the proliferation of PSC and allows their differentiation into MSCs. Treatment of PSC with Tempol, a ROS inhibitor, and Ang II reduces the cell proliferation and DNA synthesis, indicating the involvement of ROS signaling (Ahmadian et al., 2015). The presence and activation of the JAK/ STAT pathway play a crucial role in stem cell renewal. It causes p38 phosphorylation and eventually, results in the differentiation of iPSCs in the target cell. A study showed that Ang II could stimulate hypertrophy in human embryonic stem cells (hESC)-and iPSCs derived cardiomyocytes (Földes et al., 2011). However, no significant effect was observed in cell death after treatment of Ang II (200 nM) with hESC-and iPSCs derived cardiomyocytes (Nunes et al., 2017). Immunofluorescent study and RNA seq revealed the involvement of low AT 1 R expression in iPSC cardiomyocytes. Long-term Ang II incubation up-regulates AT 2 R expression to induce downstream apoptosis signaling in iPSCderived cardiomyocytes . Whereas, shortterm Ang II treatment reduces Young modulus in rat cardiomyocytes via AT 1 R. Inhibition using Rho-kinase or TGF-β1 abolished this effect (Swiatlowska et al., 2020).
Erythropoiesis is a tightly regulated process reinforced by systematic hematopoietic progenitor cells and some cohort of multipotent hematopoietic stem cells (HSCs) at its apex. HSCs form the basis of widely practiced therapies, including bone marrow transplantation and stem cell therapies (Jokubaitis et al., 2008). Chronic Ang II infusion imparts important regulatory roles on HSC proliferation, differentiation, and engraftment at the level of bone marrow (Kim et al., 2016) Flow cytometry analysis has revealed the involvement of Ang II in the regulation of hematopoiesis in HSCs and granulocyte/ monocyte progenitor cells but, not in megakaryocytes/erythroid progenitors when coculture with stromal S17 cells. (Costa et al., 2021). In addition, protein microsequencing has also reported an increase in Gr-1+/Mac-1+ cells, BB9 protein and decrease expression of Ki67+ in presence of Ang II (Jokubaitis et al., 2008;Costa et al., 2021).

CONCLUSION AND FUTURE PROSPECTS
In the last decades, there has been a noteworthy progression in the number of molecules targeting Ang II signaling pathways.
Since the discovery of Ang II, it has been characterized to be involved in various cellular activities such as proliferation, contractility, apoptosis, dysfunctions, remodeling, etc. Dysregulated Ang II signaling is considered to induce various cardiovascular diseases involving hypertension, inflammation, myocardial infarction, atherosclerosis, fibrillation, ventricular dystrophies, etc. While many ARBs are available commercially, the statistics indicate the need to look for novel mechanisms of Ang II causing cardiovascular diseases independently, which would provide a therapeutic target. The involvement of the RAS component in cardiovascular tissue and progenitor cells may regulate development and growth; and thus, allow the preparation of cardiac progenitor cells for clinical transplantation. Being a multi-functional peptide, Ang II stands amidst varied systems integrating multiple cellular signaling events that broadly have counterregulatory or opposing actions. The turmoil in the balance of such processes can lead to diverse pathologies and dysfunctions. Understanding how these processes are integrated with each other in real-time and remain a formidable challenge. Further studies with transgenics or cell-specific knockouts are needed to unravel the role of inflammatory and hypertrophic markers in Ang II-induced remodeling. Another challenge is providing evidence for working with cardiovascular progenitor cells for clinical transplantation. Further, the role of epigenetic/genetic programming impacting the cardiovascular system is an emerging area of study. Many questions still remain as well regarding paracrine, autocrine, and intracrine actions of Ang II and its interaction with receptor-associated proteins. In addition, an approach that integrates elements of metabolomics, proteomics, epigenomics, and genomics is likely to reveal novel, as yet unforeseen facet of Ang II signal transduction. Understanding diverse regulatory signaling mechanisms of Ang II can, therefore, offer better insight into various pathological states which in turn may help in designing ideal drug candidates for their management.