ROCK and PDE-5 Inhibitors for the Treatment of Dementia: Literature Review and Meta-Analysis

Dementia is a disease in which memory, thought, and behavior-related disorders progress gradually due to brain damage caused by injury or disease. It is mainly caused by Alzheimer’s disease or vascular dementia and several other risk factors, including genetic factors. It is difficult to treat as its incidence continues to increase worldwide. Many studies have been performed concerning the treatment of this condition. Rho-associated kinase (ROCK) and phosphodiesterase-5 (PDE-5) are attracting attention as pharmacological treatments to improve the symptoms. This review discusses how ROCK and PDE-5 affect Alzheimer’s disease, vascular restructuring, and exacerbation of neuroinflammation, and how their inhibition helps improve cognitive function. In addition, the results of the animal behavior analysis experiments utilizing the Morris water maze were compared through meta-analysis to analyze the effects of ROCK inhibitors and PDE-5 inhibitors on cognitive function. According to the selection criteria, 997 publications on ROCK and 1772 publications on PDE-5 were screened, and conclusions were drawn through meta-analysis. Both inhibitors showed good improvement in cognitive function tests, and what is expected of the synergy effect of the two drugs was confirmed in this review.


Introduction
Dementia refers to a significant decrease in an individual's cognitive level accompanied by memory, thought, and behavior-related disorders caused by brain damage due to injury or disease, and it hinders the patient's social function [1]. Although dementia usually occurs in the elderly, it is not an inevitable consequence of aging. It is currently the seventh leading cause of death among all diseases and a major cause of disability among the elderly worldwide. Dementia has a physical, psychological, social, and economic impact, not only on patients with dementia but also on their acquaintances, families, and society at large.
There are two main types of dementia: Alzheimer's disease (AD) and vascular dementia (VD). AD is a neurodegenerative disease and accounts for 60-70% of patients with dementia [2]. AD causes abnormal accumulation of amyloid-beta (Aβ) as amyloid plaque and tau protein and the formation of neurofibrillary tangles (NFT), resulting in gradual loss of brain function [3]. In the early stages, patients experience mild cognitive impairment, indifference, and depression [4]. As the disease progresses, disturbances in language, changes is incomplete ischemia and selective tissue necrosis, causing selective neurological necrosis due to decreased functioning of neuroglial cells and microvessels [35][36][37].

ROCK and AD
Mutation of presenilin (PSEN) was confirmed in cases of early-onset AD [54]. The APP is cleaved by both β-secretases and γ-secretase enzymes to form Aβ, and PSEN is a component of γ secretase. Therefore, mutation of PSEN leads to an increase in the Aβ42:Aβ40 ratio due to an increase in the expression of Aβ42, which promotes early Aβ deposition [55]. ROCK contributes to these secretases cleaving APP to increase Aβ production. The increase in Aβ levels further suggests a positive feedback role for ROCK, though the specific basic mechanism for this remains unclear [56].
In addition, hyperphosphorylation of tau, a characteristic of AD, seems to be associated with ROCK. Although no specific mechanism has been identified, ROCK activation activates tau kinase and inhibits tau phosphatase, increasing the expression of P-tau and oligomeric tau. It also reduces the microtubule-binding of tau and increases the formation of NFTs in neurons [57].

ROCK and Vascular Remodeling
ROCK is involved in vascular remodeling, which starts with RhoA. Downstream targets of ROCK include MLCP, LIMK, Ezrin/Radixin/Moesin (ERM) intermediate filaments, and other factors affecting intracellular processes that are important for cell contraction, movement, proliferation, and morphology ( Figure 1). RhoA binds to GTP from the Gprotein-receptor. When the cytoplasmic concentration of ROCK and Ca 2+ increases through guanine nucleotide exchange factors, the increased ROCK phosphorylates MLC [58]. The increase in MLC phosphorylation results in the contraction of the smooth muscle by combining myosin crossbridge and actin filaments. LIM phosphorylated by ROCK then phosphorylates cofilin to inhibit actin decomposition activity. Cofilin is an actin-binding protein associated with the rapid depolymerization of actin microfibers. It regulates the assembly and decomposition of actin filaments. The ERM family crosslinks the protoplasmic membrane and the actin filament to prevent actin-binding according to the folding of the ERM protein.
ROCK is also related to endothelial NOS (eNOS): it is the upstream negative regulator of eNOS, and its expression reduces eNOS expression [59]. eNOS has a protective function in the cardiovascular system due to nitric oxide (NO) production. NO catalyzes the conversion of guanosine triphosphate to cGMP by activating the enzyme soluble guanylate cyclase (sGC). This cGMP acts on vascular relaxation and contributes to vascular remodeling. NO production of eNOS inhibited by ROCK ultimately reduces the cGMP of VSMCs, thereby constricting blood vessels. In animal experiments of the VD model, an increase in ROCK expression was shown [60]. ROCK prevents dephosphorylation of phosphorylated MLC and contributes to smooth muscle contraction by activating the LIMK2/cofilin pathway and the ERM pathway. PDE-5 decomposes increased cGMP from eNOS/NO, contributing to the contraction of VSMC. cGMP: cyclic Guanosine monophosphate; eNOS: endothelial nitric oxide synthase; ERM: ezrin/radixin/moesin; GTP: guanosine triphosphate; LIMK: Lin11-Isl1-Mec3 kinase; MLC: myosin light chain; NO: nitric oxide; PDE-5: phosphodiesterase-5; ROCK: rho-associated protein kinase.
ROCK is also related to endothelial NOS (eNOS): it is the upstream negative regulator of eNOS, and its expression reduces eNOS expression [59]. eNOS has a protective function in the cardiovascular system due to nitric oxide (NO) production. NO catalyzes the conversion of guanosine triphosphate to cGMP by activating the enzyme soluble guanylate cyclase (sGC). This cGMP acts on vascular relaxation and contributes to vascular remodeling. NO production of eNOS inhibited by ROCK ultimately reduces the cGMP of VSMCs, thereby constricting blood vessels. In animal experiments of the VD model, an increase in ROCK expression was shown [60].

ROCK and Neuroinflammation
Neuroinflammation is an inflammation of nerve tissue and can begin as a response to various signals, including infection, traumatic brain damage, toxic metabolites, or autoimmune. Neuroinflammation is a common feature observed in many neurodegenerative disorders and is an important factor in neurodegenerative progression. The involvement of local innate immune responses contributes greatly to central nervous system (CNS) damage [61].

ROCK and Neuroinflammation
Neuroinflammation is an inflammation of nerve tissue and can begin as a response to various signals, including infection, traumatic brain damage, toxic metabolites, or autoimmune. Neuroinflammation is a common feature observed in many neurodegenerative disorders and is an important factor in neurodegenerative progression. The involvement of local innate immune responses contributes greatly to central nervous system (CNS) damage [61].
Activation of the RhoA/ROCK pathway increases the permeability of inflammatory factors in response to inflammatory stimuli ( Figure 2). The RhoA/ROCK pathway, through G-protein-receptors, is upregulated by chemokine-like MCP-1 and increases resolution from occludin, claudin-5, ZO-1 Ser/Thr phosphorylation, and tight junctions (TJ) to increase the barrier [62]. In addition, intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) increase inflammatory cell penetration in the CNS from RhoA/ROCK activation. Studies have shown that ROCK activation promotes neutrophil penetration in inflammation through NADPH oxidase activation and ROS generation.
to increase the barrier [62]. In addition, intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) increase inflammatory cell penetration in the CNS from RhoA/ROCK activation. Studies have shown that ROCK activation promotes neutrophil penetration in inflammation through NADPH oxidase activation and ROS generation. ROCK also directly increases the inflammatory response. Section 3.2 mentions eNOS and NO, which also inhibit chemokine and nuclear factor kappa B (NF-κB). Therefore, an increase in ROCK leads to a decrease in eNOS, resulting in an increase in the inflammatory response. In addition, the activated RhoA induces activation of p38 mitogen-activated protein kinase (p38 MAPK), which induces up-regulation of IL-4, IL-10, and INF-γ production [63,64]. ROCK2 also serves to phosphorylate the transcription factor IRF4 required for IL-17 and IL-21 generation and IL-17 T cell differentiation [65]. A study has shown that upon applying ischemic injury to the brain, the microglial proliferation contributing to neuroinflammation increases [66].
Cyclophilin A (CyPA) is a protein belonging to the immunophilin family. When ROS is induced in a hypoxic state, it stimulates CyPA secretion along with ROCK activity. CyPA secreted from VSMCs binds to basigin, a receptor outside the cell, to regulate the cell signal pathway [67] and acts as a chemical inducer for inflammatory cells [68,69]. ROCK also directly increases the inflammatory response. Section 3.2 mentions eNOS and NO, which also inhibit chemokine and nuclear factor kappa B (NF-κB). Therefore, an increase in ROCK leads to a decrease in eNOS, resulting in an increase in the inflammatory response. In addition, the activated RhoA induces activation of p38 mitogen-activated protein kinase (p38 MAPK), which induces up-regulation of IL-4, IL-10, and INF-γ production [63,64]. ROCK2 also serves to phosphorylate the transcription factor IRF4 required for IL-17 and IL-21 generation and IL-17 T cell differentiation [65]. A study has shown that upon applying ischemic injury to the brain, the microglial proliferation contributing to neuroinflammation increases [66].
Cyclophilin A (CyPA) is a protein belonging to the immunophilin family. When ROS is induced in a hypoxic state, it stimulates CyPA secretion along with ROCK activity. CyPA secreted from VSMCs binds to basigin, a receptor outside the cell, to regulate the cell signal pathway [67] and acts as a chemical inducer for inflammatory cells [68,69].

PDE-5 and AD
The relationship between PDE-5 and AD focuses on the NO pathway. The NO/sGC/ cGMP signaling pathway appears abnormally in the AD brain [80][81][82][83]. In aging wild mice, eNOS deficiency showed an increase in Aβ production [84,85], and the depletion of iNOS in AD mice with APP mutation resulted in elevated levels of Aβ and hyperphosphorylation of tau [86]. This hyperphosphorylation of tau is thought to be because Akt inhibits glycogen synthase kinase-3 beta (GSK-3β), mediating tau phosphorylation due to the activation of the P13K/Akt pathway of NO. Therefore, the decrease in NO due to PDE-5 leads to an increase in tau phosphorylation. PDE-5 is upregulated in the cerebrospinal fluid (CSF) of patients with AD, and cGMP levels are decreased [87].

PDE-5 and Vascular Remodeling
PDE-5 breaks down cGMP produced from NO/sGC/GTP signals. cGMP acts on blood vessels and contributes to vascular relaxation. When the cGMP in the VSMCs is reduced by PDE-5, the blood flow to the brain may reduce due to vasoconstriction ( Figure 1).

PDE-5 and Neuroinflammation
cGMP regulates intracellular inflammatory responses. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that contributes to the inflammatory response by recruiting monocyte, memory cells, and dendritic cells at the inflammatory site [82,88]. NO reduces the expression of MCP-1 mRNA, and the reduction of NO increases the expression of MCP-1 mRNA [89]. The regulation of the expression of MCP-1 may reduce the decomposition of IκB inhibiting NF-κB, thereby reducing the inflammatory reaction. PDE-5 may decompose cGMP in the initial steps of this process, contributing to neuroinflammation.
Astrocyte is a neuroglial cell of the brain and spinal cord. It is involved in blood-brain barrier formation and function [90], neurotransmission [91], nutrition to nerve tissue, the balance of extracellular ions, and regulation of cerebral blood flow [92]. The central immune role of astrocytes is controlled through the cGMP/PKG pathway through NO [93]. Protein kinase G (PKG) phosphorylates many targets by cGMP and is involved in functions such as smooth muscle relaxation. cGMP inhibits the expression of major histocompatibility complex II (MHC-II) derived from interferon-γ (INF-γ) in astrocytes and the expression of matrix metallopeptidase 9 (MMP-9) and tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS) [93,94]. PDE-5 can lead to neuroinflammation by increasing the expression of INF-α, MMP-9, and TNF-α derived from astrocytes in the brain by decomposing cGMP.

Inhibition of ROCK and PDE-5 Pathway for Neuroprotection
Overexpression of ROCK has been shown to cause an increased inflammatory response, increased oxidative stress, high oxidation of tau, and cognitive decline due to β-amyloid accumulation. In such a situation, ROCK inhibitors are a good choice for treating dementia. Fasudil is a typical ROCK inhibitor with potential neuroprotective effects that can cause neurogenesis and increased neuronal viability [95,96]. Inhibition of ROCK2 leads to nerve survival and axon stability [95,97]. Like Fasudil, a representative ROCK inhibitor, Y-27632 is a good option for dementia treatment. The resulting ROCK inhibition has been shown to reduce TNF-α mediated monocyte migration [98,99]. ROCK-suppressed macrophages showed reduced chemotaxis for MCP-1/CCL2. PDE-5 suppression is another possibility to improve dementia. PDE-5 inhibitors increase the cGMP concentration by blocking the cGMP decomposition of PDE-5 described above, and the increased cGMP expands blood vessels and improves blood flow through smooth muscle tissue relaxation. Typical PDE-5 inhibitors include sildenafil, vardenafil, and tadalafil. Subsequent animal studies with sildenafil demonstrated long-term retention of an inhibitory avoidance response in mice. In an in vitro study using N9 microglia, it was shown that cGMP accumulated because of regression of PDE-5 following sildenafil treatment could contribute to inhibiting microglia activation. In addition, injection of PDE-5 inhibitors into the ischemic stroke rat model with reduced cognitive function through middle cerebral artery occlusion (MCAo) improved neurological deficits and anxiogenic disorder and improved locomotion [100].

Meta-Analysis of ROCK Inhibitors and PDE-5 Inhibitors in Animal Experiments
In this study, a meta-analysis was performed to investigate the relationship between cerebrovascular disease and drug effects of ROCK and PDE-5 inhibition in animal models. A meta-analysis is a quantitative, formal, epidemiological study design used to systematically assess previous research studies to derive conclusions about that body of research. It can be performed when there are several scientific studies addressing the same problems, and each study reports measurements that are expected to be somewhat error-prone. The efficacy of ROCK inhibitors and PDE-5 inhibitors as drug treatments for dementia in animal models is analyzed and presented here.  (Table 1), and data were extracted from each study after completing the search.

Data Extraction
The Morris water navigation task, also known as the Morris water maze (MWM), is a behavioral technique mostly used with rodents [124]. It is predominantly used in behavioral neuroscience to study spatial learning and memory. The basic procedure involved in MWM is that the mouse or rat is placed in a large circular pool and must find an invisible or visible platform that allows it to escape the water using various cues. The time it takes to escape is measured, and the maze is divided into quarters to help measure how long the animal stays in the target area. Animal models of neurotrauma, cerebrovascular disease, developmental disorders, metabolic disorders, AD, and other disorders with neurocognitive disorders and cognitive complications have been demonstrated to differ from healthy models using MWM [125][126][127][128][129][130][131][132][133][134][135][136][137][138][139][140]. In addition, as an evaluation of neurocognitive treatment, it was confirmed that the performance of MWM improved following behavior, pharmacological, and neurosurgical interventions [141][142][143][144][145][146].
Two reviewers (D.H.L. and J.S.O.) independently extracted data according to a predetermined data extraction form. Duplicate data was removed using Endnote. Features extracted from each study include the first author's name, publication year, pathology induction method of the disease group, drugs used in the experimental group receiving pharmacotherapy, gender, age, pharmacotherapy method, drug dosage, disease classification (AD or VD), MWM-mean value of time spent in the target quadrant (TSTQ), SD values (%), and mice participating in the experiment (Tables 2 and 3).

Data Analysis
Meta-analysis was performed using a random effect model. The results were presented according to the 95% confidence interval (CI). A heterogeneity test was performed using the Cochran Q test, and a publication bias test was performed using the Egger's test. Statistical analysis was conducted using Revman (version 5) software.
Because of the high heterogeneity, each group was sub-classified into a subgroup, and a meta-analysis was performed.

Results
A total of 997 publications on ROCK inhibitors were identified through the search formula. According to the screening criteria, 11 publications were finally selected ( Figure 3) after (1) removing duplicates; (2) removing articles with an irrelevant title; (3) removing articles with an irrelevant abstract (excluded if it was based only traditional medicine, herb extracts, alkaloids, flavonoids, no dementia model animal or patient, no cognition assessment, no ROCK inhibitors, or there was no clinical trials); (4) evaluating the abstract and full text for eligibility; (5) excluding articles that effect of ROCK inhibitors that failed to demonstrate any effects in behavior test; and (6) articles containing quantitative data of TSTQ of MWM test for meta-analysis. Of these, nine studies were on AD and two on VD. For PDE-5 inhibitors, 1772 publications were searched and screened according to the screening criteria ( Figure 4). Finally, 12 publications were selected according to the screening criteria. Nine of these focused on AD and three on VD. The date of the most recent publication was 31 July 2021 [147].   (3) According to the eligibility, publications that had no therapeutic effect in the behavioral experiment were excluded. (4) Full-text articles that were not available for meta-analysis, that had no animal, cognition, or Morris water maze test, were excluded.
A total of 414 mice participated in the 23 finally selected studies. Both male and female mice were included in these studies. Their age was widely distributed from 8 weeks to 18 months (Table 2).
Of these, 330 (79.8%) mice had AD, and 84 (20.2%) mice had VD. Methods that induced AD included drug administration and genetic mutations. The intracerebroventricular streptozotocin (ICV-STZ) method, which induces AD in animals without transformation, shows mitochondrial abnormalities [148], decreased glucose use, increased tau phosphorylation, and neurochemical changes in the brain, such as the 3xTG-AD mouse [149]. AD was induced in 64 (19.4%) mice through ICV-STZ injection. In addition to drug induction, there is a model that mimics human AD with genetic mutations. Further, 235 (2) Publications with unrelated titles and abstracts were excluded. (3) According to the eligibility, publications that had no therapeutic effect in the behavioral experiment were excluded. (4) Full-text articles that were not available for meta-analysis, that had no animal, cognition, or Morris water maze test, were excluded.
A total of 414 mice participated in the 23 finally selected studies. Both male and female mice were included in these studies. Their age was widely distributed from 8 weeks to 18 months (Table 2).
Of these, 330 (79.8%) mice had AD, and 84 (20.2%) mice had VD. Methods that induced AD included drug administration and genetic mutations. The intracerebroventricular strep-tozotocin (ICV-STZ) method, which induces AD in animals without transformation, shows mitochondrial abnormalities [148], decreased glucose use, increased tau phosphorylation, and neurochemical changes in the brain, such as the 3xTG-AD mouse [149]. AD was induced in 64 (19.4%) mice through ICV-STZ injection. In addition to drug induction, there is a model that mimics human AD with genetic mutations. Further, 235 (71.2%) mice developed AD due to gene mutations. APP is a precursor molecule that produces Aβ and is a major component of amyloid plaque found in the brain of AD patients. PSEN-1 (PS1) plays an important role in Aβ generation by cleaving APPs and regulating their activity. Tg2576 and J20 induced mutations in these APPs, and APP/PS1 induced mutations in both APP and PS1 resulted in AD.
Mice with conditions imitating VD-induced arteriosclerosis with cholesterol crystals, ischemic stroke with bilateral common carotid artery occlusion (BCAO), or bilateral common carotid artery ligation had hypoxic damage to the brain.
ROCK inhibitors for pharmacological treatment of dementia (11 out of 23) include Fasudil and Y-27632. Ganoderma lucidum triterpenoids, Edaravone, and Clitoria ternatea have also been verified to inhibit the ROCK pathway. PDE-5 inhibitors (12 out of 23), including sildenafil, tadalafil, vardenafil, CM-414, and KJH-1002 have also been verified in each publication. The drugs were either stereotaxic, orally-administered, injected into the left lateral ventricle, administered through gavage, or through intraperitoneal or intracerebral vascular injection. Of all mice, a total of 205 (49.5%) mice were treated with either ROCK inhibitors or PDE-5 inhibitors, 98 (23.7%) of which were treated with ROCK inhibitors, and 107 (25.8%) were treated with PDE-5 inhibitors.
Considering that the scales of all studies are not the same and the heterogeneity is high, the analysis was divided into standardized mean difference (SMD, Figure 5) and mean difference (MD, Figure 6). their activity. Tg2576 and J20 induced mutations in these APPs, and APP/PS1 induced mutations in both APP and PS1 resulted in AD.
Mice with conditions imitating VD-induced arteriosclerosis with cholesterol crystals, ischemic stroke with bilateral common carotid artery occlusion (BCAO), or bilateral common carotid artery ligation had hypoxic damage to the brain.
ROCK inhibitors for pharmacological treatment of dementia (11 out of 23) include Fasudil and Y-27632. Ganoderma lucidum triterpenoids, Edaravone, and Clitoria ternatea have also been verified to inhibit the ROCK pathway. PDE-5 inhibitors (12 out of 23), including sildenafil, tadalafil, vardenafil, CM-414, and KJH-1002 have also been verified in each publication. The drugs were either stereotaxic, orally-administered, injected into the left lateral ventricle, administered through gavage, or through intraperitoneal or intracerebral vascular injection. Of all mice, a total of 205 (49.5%) mice were treated with either ROCK inhibitors or PDE-5 inhibitors, 98 (23.7%) of which were treated with ROCK inhibitors, and 107 (25.8%) were treated with PDE-5 inhibitors.
Considering that the scales of all studies are not the same and the heterogeneity is high, the analysis was divided into standardized mean difference (SMD, Figure 5) and mean difference (MD, Figure 6).
Mice, who received all treatments, had an average of 1.83% SMD (95% CI 1.43-2.24, I 2 64%) more cognitive improvement than before treatment. Among them, mice treated with PDE-5 inhibitors had 1.80% SMD (95% CI 1. 25   . This forest plot shows an analysis of the difference between the experimental group and the control group of each subgroup by standardized mean difference (SMD). Because of the high heterogeneity, each group was analyzed by dividing it into subgroups. SD: standard deviation. Mice, who received all treatments, had an average of 11.78% MD (95% CI 9.66-13.89, I 2 73%) more cognitive improvement. Among them, mice treated with PDE-5 inhibitors had 10

Conclusions
Dementia, which causes cognitive impairment, is one of the main causes of death and affects the families of many patients globally. The main causes of dementia are AD and VD, and its treatment is difficult. Many mechanism studies on the treatment of dementia Mice, who received all treatments, had an average of 1.83% SMD (95% CI 1.43-2.24, I 2 64%) more cognitive improvement than before treatment. Among them, mice treated with PDE-5 inhibitors had 1.80% SMD (95% CI 1.25-2.34 I 2 61%) cognitive improvement before treatment, while mice treated with ROCK inhibitors had 1.87% SMD (95% CI 1.25-2.49 I 2 66%) cognitive improvement than before treatment. The biggest SMD improvement among publications with ROCK inhibitor treatment was found in the report of Yun  88-43.70), as re-ported by Manish AD ROCKI 2018. On the other hand, the PDE5I treatment with the smallest MD difference was YU AD ROCKI 2020, which reported an improvement of 5.67% (95% CI 1.67-9.67).

Conclusions
Dementia, which causes cognitive impairment, is one of the main causes of death and affects the families of many patients globally. The main causes of dementia are AD and VD, and its treatment is difficult. Many mechanism studies on the treatment of dementia have been performed. We focused on studies of ROCK and PDE-5. ROCK and PDE-5 contribute to AD deterioration through PS1 mutation and tau hyperphosphorylation, respectively; cause damage to the brain due to hypoxia, induced by decreased cerebral blood flow due to vasoconstriction; and contribute to increased pro-inflammatory marker levels and immune cell migration. Therefore, ROCK and PDE-5 inhibitors are receiving significant attention as pharmacological treatments for dementia. Improvement of the cGMP pathway and an increase in the cognitive function of mice following ROCK or PDE-5 inhibition were confirmed in animal and in vitro experiments. In addition, the MWM-TSTQ (%) results of animal models were compared based on the SMD and MD through meta-analysis, confirming that both ROCK inhibitors and PDE-5 inhibitors helped improve cognitive function.
Therefore, the results of this analysis expect synergistic treatment effects for the combined administration of both drugs. Both ROCK and PDE-5 inhibitors showed good effects on improving cognitive impairment, and many factors share both mechanisms. ROCK suppresses eNOS and PDE-5 suppresses NO, leading to a downward adjustment of NO/cGMP. And they share several factors that can affect neuroinflammatory responses, such as MCP-1, NF-kB, and VCAM-1. So we conclude that the combined administration of both inhibitors is worth studying in anticipation of the treatment effect of synergy.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing does not apply to this article.

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