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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: [email protected]
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.