Sonic hedgehog signaling activation promotes cardioprotective strategies

Himanshi Khera1, Anupam Awasthi1, Sidharth Mehan*1

Department ofPharmacology,

ISF College of Pharmacy, Moga-142001, Punjab, India

Corresponding Author*1

Dr. Sidharth Mehan

Ph.D, M.Pharm, DNHE, CFN, CNCC

Associate Professor,

Department of Pharmacology,

ISF College of Pharmacy, Moga-142001, Punjab, India

Mail: sidh.mehan@gmail.com

Phone: +91-8059889909, +91-9461322911

Abstract

Hedgehog pathway plays a crucial role in the neovascularisation and angiogenesis during the

embryonic stage in humans. Three genes of hedgehog protein have been isolated from humans

are Sonic hedgehog, Desert hedgehog and Indian hedgehog gene. Two G-protein coupled

receptors identified in sonic hedgehog pathway served as patched receptor and smoothened

receptor. Particularly sonic hedgehog gene plays a versatile role in cellular homeostasis and can

be a novel therapeutic target in the prevention of cardiovascular disorders. Further various sonic

hedgehog modulators have been reported and working as a futuristic drug molecules in the

modulation of cardiovascular dysfunctions. However, there was limited literature availability that

has summarized the possible mechanism of targeting Sonic hedgehog signaling pathway. Thus,

the present review is aimed at exploring the role of targeting sonic hedgehog protein signaling

and modulators as well as to enlighten that how targeting sonic hedgehog protein involve in the

amelioration of atherosclerosis, ischemic heart diseases, vascular endothelial dysfunction, heart

failure and congenital heart diseases.

Keywords: Sonic hedgehog, Cardiovascular disorders, Angiogenesis, Vascular endothelial

dysfunction, Hedgehog pathway, Ischemic heart disease.

Abbreviations:

HhHedgehog

ShhSonic hedgehog

DhhDesert hedgehog

IhhIndian hedgehog

SmoSmoothened

PtcPatched

SuFu Suppressor of fused

mAbMonoclonal antibodies

CVDsCardiovascular disorders

LDLLow density lipoprotein

PI3-K/AktPhosphoinositide-3-kinase–protein

kinase B/AktiNOS

Inducible nitric oxide synthaseVEGF

Vascular endothelial growth factor

1. Introduction

During past decades, members of hedgehog (Hh) family of intercellular signaling proteins have

been shown to play an important role in maintaining the tissue development during

embryogenesis, neuronal development, vascular proliferation, differentiation and angiogenesis.

Specific mutation of Hh gene in Drosophila melanogaster has also been associated with various

disorders such as cyclopia, defects in ventral tube, somite, foregut patterning, and severe distal

limb malformation and even failure of lung branches [1]. The name Hh has emerged from the

short and spiked phenotype of the cuticle of the Hh mutant Drosophila melanogaster larvae.

Three genes namely sonic hedgehog protein (Shh), Desert hedgehog protein (Dhh) and Indian

hedgehog protein (Ihh) homologues to Drosophila melanogaster has been identified in humans

with distinct functional properties. Dhh and Ihh has prominent role in spermatogenesis,

pancreatic cell proliferation, bone formation, coordinating growth and differentiation of

chondrocytes in the development of endochondral skeleton and regulation of mammalian gut

[2]. While, Shh have been identified as a most potent Hh ligand during embryonic development,

adult tissue development and maintenance of tissue polarity. Specifically, Shh has been found to

play an essential role in regulation of pattern in somite, neuronal tube, limb as well as growth

and morphogenesis of various organs like hair, tooth, lung and gastro-intestinal tract.

Dysfunctioning of Shh-signaling pathway has also been reported to play a dominant role in

cardio-vascular disorders including atherosclerosis, ischemic heart diseases, endothelial

dysfunction and hypertension [3].Therefore, pharmacological modulation of the Shh-signaling

pathway could be a therapy of interest in variety of cardio-vascular disorders. Till date, there is

no information available in research database to review and targeting the sonic hedgehog-

signaling pathwayin concern with cardiovascular dysfunctions. Therefore, purpose of this review

is to explore and justify the future perspective role of Shh-signaling pathway in the field of

cardio-vascular disorders.

2. History of Hedgehog-Signaling Pathway

In 1980, Nusslein-Volhard and Wieschaus has firstly indentified a gene during the screening of

Drosophila melanogaster that affects the pattern of larval cuticle and concluded that these

phenotypic changes are due to the mutation in the Hh gene [4]. In 1990s, the Drosophila

melanogaster gene was first cloned and found to include three exons which would encode a 421-

(or 471-) amino acid polypeptide with a long hydrophobic stretch. In 1993, Hh gene was cloned

in various vertebrates like Mus musculus, Daniorerio and Gallus gallus. Later on in 1995, the Hh

gene was finally cloned in Homo sapiens. Drosophila melanogaster has one Hh gene but the

mammals have three paralogoues Hh genes namelyas Shh, Ihh and Dhh genes. It has been

reported that Shh and Ihh genes are closely related to each other but the gene of the Dhh is

closely related to the Hh gene of the Drosophila melanogaster. Firstly, in 1995, genes of the Shh

and Ihh were cloned in humans and, later in 1996 gene of the Dhh was cloned [5].

3. Synthesis, Structure and Translocation of Sonic Hedgehog Protein

Shh has been initially translated as a 45KDa precursor protein with 439 amino acids. Shh protein

is having two different regions; one is amino terminal domain named as hedge domain and

second is carboxy terminal domain named as hog domain (Figure 1). It has been found that,

amino-terminus domain act as a signaling domain and carboxy terminal domain has auto-

catalytic properties and also contain hint module. Specifically, the hint module present in the hog

domain has auto-catalytic properties [6]. Following the translation of the Shh protein, it gets

translocated to the endoplasmic reticulum lumen, where it gets processed in cholesterol

dependent manner. Finally, it gets cleaved between Gly174 and Cys175 to generate an amino

terminal as a 19KDa fragment and carboxy terminal as a 25KDa fragment. The auto catalysis of

Shh protein occurs generally follows two step mechanisms. In the first step, carbonyl group of

the Gyl174 amino acid attacked by the sulfhydryl group of the Cys175 amino acid and results in

the formation of a thio-ester intermediates. In the second step, thio-ester linkage has been found

to attack by the 3β-hydroxyl group of a cholesterol molecule finally resulting in formation of an

ester linkage between a cholesterol moiety in the presence of cholesterol transferase and the

carboxy-terminal of Gly174 of the amino terminal fragment [7]. After the addition of cholesterol

moiety to the carboxy terminal of the amino terminal fragment of Shh protein, palmitoyl group

has been found to be added to the amino terminal. Palmitoylation of Shh protein catalyzed by

Skinny hedgehog acetyl transferase. Interestingly, it has been found that amino terminal cysteine

lacked Shh protein are cholesterol modified but remain as un-palmitoylated. Addition of the

cholesterol moiety has been found to play a dominant role in controlling the secretory regulation,

long-term activity and signaling modifier of the Shh protein. Specific blockade of the cholesterol

synthesis during the early embryogenesis was found to be associated with various birth defects

likecyclopia, midline defects and variations of holoprosencephaly. In addition to this,

palmitoylation has been found to increase the inductive potency of the Shh protein by playing an

essential role in binding of Shh protein to its receptor proteins [8].

The active fragment of Shh protein contains 169 amino acids, that has been reported to allow

Shh to form a symmetric structure that is comprise of 3 α-helices and 8 β-sheets [9]. It has been

concluded from the series of X-ray crystallography studies that the activated Shh protein

possesses two different metal ions Ca2+ and Zn2+(Figure 2) [10]. Interestingly, the Shh contains

two Ca2+ ion that are bound in loop regions by amino acids such as Glu91, Glu127, Asp96,

Asp130 and Asp132. Shh contains Zn2+ ion coordinated by His141, His183, Asp148 and water

molecule that bridged the Zn2+ ion with Glu177 [9]. Further, Ca2+ and Zn2+ ion has been well

reported to present in the different sites of activated Shh protein whereas, Zn2+ ion predominantly

present on the non-catalytic site and Ca2+ reportedly present on the active site of the activated

Shh protein [11]. Interestingly, these two metal ions are reported to play an essential role in Shh

protein folding and its interaction with the receptor [12]. Indeed, activated Shh protein has two

lipid molecules out of which one is cholesterol present on the carboxy terminal and other

palmitic acid on amino terminal domain. Due to the presence of dual lipid molecules in activated

Shh protein they remain anchored to the cell membrane and also promote the clustering of these

activated Shh-signaling proteins [13]. Further, cholesterol mediated clustering has been found to

promote the interaction of positively charged residues within a conserved Cardin-Weintraub

motif which is present in the activated Shh-signaling protein in association with heparin sulfate

proteoglycans [14]. Scube2 (signal peptide, cubulin domain, epidermal-growth-factor-like

protein 2), a secreted glycoprotein has been implicated in the release of Shh protein from the cell

membrane of the cell to which they are anchored through ectodomain shedding [15]. The

ectodomain shredding of Scube2 protein is highly dependent on the CUB domain. Further, it was

found that membrane protein “Dispatched” has played an essential role along with Scube2

protein in the release of activated Shh-signaling protein from the cell membrane finally results in

the release of clustered activated Shh-signaling protein at the site of action [16].

4. Signal Transduction of Sonic Hedgehog Pathway

Shh protein activation act as a ligand on sterol sensing domain of the patched (Ptc) receptor

proteins specially in cilia of eukaryotic cells [17]. Cilia, a microtubule-based organelle or tail like

projections present on the eukaryotic cells has been reported to be involved in the sensing

chemical and mechanical signals. Interestingly, all the proteins which play a role in Shh

signaling are enriched in the cilia of the cells [18]. The binding of the activated Shh protein has

been highly affected by the cell-surface regulator proteins. Cdo and Boc, the two cell surface

regulatory proteins which enhance the binding of the activated Shh protein to the Ptc receptors

[19]. In addition to this, growth arrest-specific-1 protein has also been found to increase the

binding affinity of the Shh protein to Ptc receptors. However, in the contrary, Hedgehog-

interacting protein-1, a inhibitory cell-surface protein interfere with the binding of the Shh

protein to the Ptc receptors [20].

Ptc receptor proteins are 12-transmembrane proteins which are homologues to the G-protein

coupled receptors. Further, two types of Ptc receptors has been reported to present in humans are

Ptc1 and Ptc2 [21]. Indeed, the Ptc1 has a privilege of being most extensively studied Shh

receptor; the Ptc2 has shown similar activity as of Ptc1 in terms of binding to the activated Shh

protein and also in terms of its interaction with other signaling proteins [22]. However, it has

been concluded that all the actions of activated Shh are exerted through the Ptc1 receptors. Ptc

receptors also called as negative receptors because in the absence of the activated Shh protein it

represses the transcription of the Shh dependent pathways [23].

Once the Ptc receptor get activated by Shh protein complex, it further activate a signal

transducing protein named smoothened (Smo) receptors, a member of the Frizzled family of

seven-transmembrane domain proteins [24]. Smo receptors has been classified in the category of

the positive receptors, because once they are activated they promote the transcription of the Shh

dependent pathways. Once the Shh ligand interacts with Ptc receptors, the Ptc receptors complex

with activated Shh protein get internalized towards to cytosol from the outer membrane and lost

its activity immediately [25]. In the contrary, the Smo receptors get transported from the cytosol

towards the ciliary membrane and gain their activity. Interestingly, in the absence of Shh

binding, Ptc was found to unphosphorylate Smo and translocate it from the ciliary membrane to

cytosol by endocytosis and finally get degraded by lysosomes. Once Shh bound to Ptc, activated

Ptc complex hyperphosphorylate the Smo protein and completely obstruct its lysosomal

degradation and endocytosis. That finally results in the regaining of the activity of Smo protein

on the transmembrane of cilia of eukaryotic cells [26]. Once Smo receptor protein is activated by

Shh bound Ptc complex, there are series of proteins that has been reported to achieve activation

[27]. Many proteins like COS2, suppressor of fused (SuFu), Kif7 and fused have been identified

in the Drosophila melanogaster [28]. However, in mammalian cells, there are two proteins Rab23

and tectonic, and these proteins have been reported to negatively regulate the downstream of Ptc-

Smo signaling. Finally, the activation of Smo-dependent series of proteins leads to the activation

of Cubitus interrupts or Gli family of proteins. Gli is a zinc-finger protein, which finally regulate

target genes by binding to the specific binding site present on the promoter region of the target

genes [29]. In vertebrates, Gli having three isoforms Gli1, 2 and 3 whereas, Gli1 is the only full

length transcriptional activator that is present in the vertebrates and, Gli2 and 3 could be either

positive or negative regulator of Shh-signaling mediated transcription (Figure 3).

5. Targeting sonic hedgehog signaling in various cardiovascular disorders

Cardiovascular disorders (CVDs) are group of dysfunctions associated with heart and blood

vessels. It has been reported that CVDs are one of the leading cause of mortality worldwide and

are responsible for approximately 17.7 million deaths in 2015. The number of deaths associated

with CVD’s has decreased gradually in the last decades in western countries. However, CVD’s

are the major cause of morbidity and mortality in developing countries [30].

Shh-signaling pathway was found to have role in developing organisms remains active during

the adult stage and plays a dominant role in maintaining cellular physiology and homeostasis.

Specifically, Shh signaling pathway has a prominent role in promoting adult cell proliferation,

neuronal plasticity, neovascularisation, angiogenesis and organ repair [31]. Modulation of Shh

signaling pathway has been found to be beneficial in various disorders such as cancer,

neurodegeneration, osteogenesis, renal ischemia, gastric abnormalities, ocular dysfunctions and

numerous CVDs including coronary artery diseases, hypertension, atherosclerosis and

myocardial infarction.

5.1Targeting sonic hedgehog signaling in atherosclerosis

Atherosclerosis is a progressive disease resulted from hyperlipidemia that finally results in the

accumulation of lipids and fibrous elements in the large arteries [32]. Atherosclerosis has been

found to be a most prevailing CVD and clinical manifestations associated with atherosclerosis

are heart attack, stroke, gangrene of the extremities, cerebrovasuclar and peripheral vascular

disease [33]. Low density lipoprotein (LDL) has a prominent role in the plaque formation during

atherosclerosis. Previously, it was hypothesized that LDL performs a dominant role in Shh

transport throughout mammalian blood stream [34]. Since, it is well established that LDL is a

bad cholesterol and helpful in transportation of cholesterol from periphery to the body [35]. Shh

protein present with the LDL molecules could have some role in progression of atherosclerotic

plaque [36]. After trapping of LDL molecules at the site of injury, Hh molecules stimulates the

series of events in the form of proliferation and migration of smooth muscle cells,

revascularization, chemotaxis and T-cell activation [37;3]. In addition to this, Ptc receptors were

found to expressed in activated T-lymphocytes [38]. Further, Shh signaling pathway could

trigger the various events during the progression of atherosclerotic plaque formation. More

specifically, Shh-signaling pathway control the expression of various downstream proteins

mainly transforming growth factor-β and bone morphogenetic proteins involved in the

pathogenesis of atherosclerosis. It has been found that upregulation of Scube2 protein plays a

dominant role in progression of atherosclerosis [39]. The Scube2 protein was diffusely

distributed in intima of arteries which were affected by the accumulation of lipids and fibrous

elements during atherosclerotic events [40]. Interestingly, the increased expression of Scube2

could be directly correlated to the release of activated Shh-signaling protein at the site of injury

during atherosclerotic plaque formation and finally responsible for the progression of the

atherosclerosis.

5.2 Targeting sonic hedgehog signaling in ischemic heart diseases

Ischemic heart diseases also known as coronary artery diseases caused by an imbalance between

supply of coronary blood flow to the heart that finally results in group of dysfunctions like

primary cardiac arrest, angina pectoris, myocardial infarction, heart failure and cardiac

arrhythmias (Figure 4). The fundamental defect in ischemic heart diseases is not only associated

with decreased blood supply but also play a prominent role in reduction of nutrient supply and

oxidative stress mediated toxicity in heart muscles [41]. Angiogenesis and remodeling of

existing blood vessels in order to compensate the decreased blood flow in areas of the heart

affected by ischemic heart diseases. Therefore, promoting the angiogenesis and remodeling

process in the existing blood vessels by administration of xenobiotic could be beneficial to cure

various types of ischemic heart diseases.

Mounting evidences suggested that the pharmacological modulation Shh-signaling pathway has a

potential therapeutic role in providing cardioprotection during ischemic heart diseases [42].

However, on the contrary, specific blockade of the Shh-signaling cascade lead to loss of

coronary blood vessels that finally performs a series of events starting from local tissue hypoxia,

cardiomyocyte death and heart failure [43].

Activation of Shh-signaling pathway has been reported to activate phosphatidylinositol 3-kinase

(PI3-K)/Akt pathway that results to increase production of nitric-oxide ultimately leads to

perform cardio-protection during ischemic heart diseases [44]. Increase production of nitric

oxide responsible for the modulation of inward potassium channels by reducing the duration of

cardiac action potential [42]. Specific activation of hypoxia-inducible factor-α during ischemia

was found to activate Shh-signaling cascade in a Gli-dependent and independent manner that

finally results in facilitation of angiogenesis [45].

Figure 4: Role of ischemia in myocardial damaging

Further, Shh-signaling dependent up-regulation of angiogenic genes such as netrin-1, iNOS,

VEFG and angiopoietins which involve in the initiation of angiogenesis [46]. Recently, it was

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confirmed that Shh-signaling plays a critical role in the maintenance of coronary

microvascularization [47]. Further, specific inhibition of Shh-signaling in mouse heart associated

with serious heart complications like coronary vessel dropout, tissue hypoxia, cardiomyocyte

apoptosis and finally death of animal [43]. In addition to this, directly increasing the expression

of Shh in cardiac cells associated with elevation of microvascularization during myocardial

infarction and specific inhibition of Shh-signaling pathway remarkably suppressed the

erythropoietin-induced protective mechanisms in infracted heart [48]. Further, SAG(Hh-Ag1.3)

has shown increased capillary density, decreased myocardial infarction and improved heart

function in diabetic mice associated with severe cardiac complications [49].

5.3Targeting sonic hedgehog signaling in vascular endothelial dysfunction

Endothelial cells present in the inner lining of blood vessels that has been reported to play a

supreme role in maintaining the smooth muscle tone [50]. The endothelial cells maintain the

smooth muscle tone by releasing both factors, endothelial contracting (endothelin-1,

vasoconstrictor prostanoids, angiotensin-II and superoxide anions) and relaxing (nitric oxide and

prostacyclin) [51]. Under physiological conditions, the balanced release of contracting and

relaxing factors has been found to maintain the vascular muscle tone [52]. Under pathological

conditions, imbalance between contracting and relaxing factors could be responsible for various

cardiovascular complications such as hypertension, diabetes mellitus, heart failure and coronary

artery diseases [53].

Mounting evidences suggested the potential role of targeting Shh-signaling pathway in

maintaining the vascular muscle tone by promoting the release of endothelial relaxing factor

named nitric oxide [54]. PI3-kinase and ERK-signaling pathway could be involved in nitric

oxide dependent maintenance of vascular tone [55]. Furthermore, activation of Shh-signaling

pathway has been found to show powerful anti-oxidant effects during vascular endothelial cell

injury [56; 57]. Shh-signaling pathway has been found to maintain the vascular endothelial

integrity of pulmonary artery ring in patients addicted to smoking through VEGF-dependent

manner. Specific inhibition of Shh-signaling pathway by GANT-61 was found to significantly

reduce the expression of VEGF and thus providing with the strong evidence of the role of Shh

signaling pathway in maintaining the vascular smooth muscle tone [58].

5.4Targeting sonic hedgehog signaling in heart failure

Heart failure is a multi-factorial progressive systemic diseases result from coordinated complex

compensatory mechanisms such as excessive volume over-load, increased adrenergic activities

and circulation redistribution during cardiac injury [59]. Over activation of compensatory

mechanisms in order to maintain the metabolic demand of the body leads heart towards serious

complications finally results in heart failure [60]. As earlier discussed that, Shh ligand expressed

in the adult heart and reported to play a dominant role in maintaining the homeostasis [61]. It has

been found that tissue-specific removal of Smo receptor protein in the adult mouse leads to

ventricular failure and even death of the animal [43]. Previous studies suggested the essential

role of Shh-signaling pathway in promoting neovascularization and angiogenesis in the adult

heart [62]. Since, both the size and function of the heart are angiogenesis dependent and

disruption of the angiogenesis during heart failure has been reported to protect the heart from

atrophy and failure [63]. As Shh-signaling pathway has a role in promoting neovascularization

and angiogenesis. Therefore, specific agonism of the Shh-signaling pathway could worsen the

condition of the heart during heart failure. However, further studies are required in this direction

to decipher the exact role of Shh-signaling pathway in heart failure.

5.5 Targeting sonic hedgehog signaling in congenital heart disease

The heart is the first organ that forms during embryogenesis to ensure proper distribution of

oxygen and nutrients during developmental stages [64]. Organogenesis of the heart is a complex

process which includes heart tube formation, looping, chamber septation and maturation. Any

abnormality occurs during the process of the heart formation comes under congenital heart

disease (65).It has been found that Shh-signaling pathway play an essential role in the

development of the heart during embryonic stage. Specific loss of Shh-signaling pathway was

found to be associated with various cardiac defects during embryogenesis are ventricular

hypoplasia, septation defects and outflow tract shortening [66]. Thus, describing the strong

evidence of the essential role of Shh-signaling pathway in cardiac outflow septation.

In addition to this tissue specific removal of Hedgehog pathway has concluded that Shh-

signaling pathway play a dominant role in cardiac neural crest, second heat field and in dorsal

mesocardium was found to be required for atrioventricular septation [67]. Similarly, specific

inhibition of Shh-signaling pathway in embryos of chick has been associated with various

congenital heart defects such as pulmonary atresia and stenosis as well as persistant

truncusarteriosus [68]. In the same line, mouse deficient in Smo-receptor protein has serious

congenital heart defects associated with abnormal heart morphogenesis, decreased heart size and

delayed initiation of Nkx2-5 expression, a pre-cardiac marker [69]. Specific mutation to the

GATA-4, a zinc finger protein act up-stream to results in the Hh-signaling pathway malfunction.

Finally, mutated GATA-4 dependent altered Hh signaling pathway has been found to play a role

in pathogenesis of congenital heart disease [70].

6. Conclusion

Hedgehog pathway plays a vital role in embryonic development and also in repair mechanism.

Due to an over expression of Shh protein there can be positive as well as negative effects on

body. It can be taken in accounts either it can induce or prevent the various physiological

dysfunctions. Detailed concept about the Shh structure, synthesis, transport and signal

transduction have emerged out to be very beneficial, as it makes it feasible to fabricate the newly

targeted drugs which directly interact with the sonic hedgehog pathway. It has been observed

that cardiovascular disorders are responsible for increased mortality rate. Therefore, targeting

sonic hedgehog pathway can be treated as novel therapeutic strategy for the prevention of

cardiovascular disorders. Literature has documented that the involvement of Shh modulators are

beneficial in case of angiogenesis and neovascularization, and it is feasible to treat vascular

endothelial dysfunction and heart related complications to much extent. Although much of the

knowledge has been gathered but here, we tried to justify and explore the sonic hedgehog

pathway in concern with relevant molecular targets associated with cardiovascular disorders.

Acknowledgement

The authors express their gratitude to Chairman, Mr. ParveenGarg and Director, Dr. G.D.Gupta,

ISF College of Pharmacy, Moga (Punjab), India for their great vision and support. Authors are

really thankful to Dr. Anoop Kumar, Department of Pharmacology, ISF College of Pharmacy,

Moga-142001, Punjab, India for valuable support and encouragement.[2

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Reviewer A:

The topic can be supported by lots of datas. references, figures etc. But in this article it not like this.

Answer: Supported data with relevant references has been added as well relevant new figure has been added in this review as per need.

Reviewer B:

1. English is OK, but another revise for grammatical and typo corrections is needed. During past decades, members of hedgehog (Hh) family of intercellular signaling proteins have been shown to play an important role in maintaining -- why passive form?

Answer: Grammatical and typo mistakes has been corrected as per guidance

2) References should be in []. not ().

Answer: Has been replaced with corrected format

3) I don't see numerical results of your work. Add a new section to describe experimental results.

Answer: respected, as per review article requirement, our manuscript has been drafted in that particular review form.

4) No related references from the journal have been cited.

Answer: Relevant references have been added.

5) Cite sample related works: 10.1007/s00521-017-2953-4 10.1007/s00521-018-3489-y 10.1007/s00521-017-2953-4

Answer: As per review article requirement, our manuscript has been drafted in that particular review form.

6) Sections 2 and 3 are not sound enough.

Answer: Respected, more addition has been included as per requirement

7) Structure of abstract is not correct according to the journal format.

Answer: Abstract has been modified as per journal format.