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EndMT Regulation by Small RNAs in Diabetes-Associated Fibrotic Conditions: Potential Link With Oxidative Stress

Frontiers in Cell and Developmental Biology

May 2021
9

DOI:10.3389/fcell.2021.683594

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CC BY 4.0

Authors:

Roberta Giordo

University of Sassari

Hilda Allam

University of Sharjah

Salah Abusnana

University Hospital Sharjah

Show all 8 authorsHide

Diabetes-associated complications, such as retinopathy, nephropathy, cardiomyopathy, and atherosclerosis, the main consequences of long-term hyperglycemia, often lead to organ dysfunction, disability, and increased mortality. A common denominator of these complications is the myofibroblast-driven excessive deposition of extracellular matrix proteins. Although fibroblast appears to be the primary source of myofibroblasts, other cells, including endothelial cells, can generate myofibroblasts through a process known as endothelial to mesenchymal transition (EndMT). During EndMT, endothelial cells lose their typical phenotype to acquire mesenchymal features, characterized by the development of invasive and migratory abilities as well as the expression of typical mesenchymal products such as α-smooth muscle actin and type I collagen. EndMT is involved in many chronic and fibrotic diseases and appears to be regulated by complex molecular mechanisms and different signaling pathways. Recent evidence suggests that small RNAs, in particular microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are crucial mediators of EndMT. Furthermore, EndMT and miRNAs are both affected by oxidative stress, another key player in the pathophysiology of diabetic fibrotic complications. In this review, we provide an overview of the primary redox signals underpinning the diabetic-associated fibrotic process. Then, we discuss the current knowledge on the role of small RNAs in the regulation of EndMT in diabetic retinopathy, nephropathy, cardiomyopathy, and atherosclerosis and highlight potential links between oxidative stress and the dyad small RNAs-EndMT in driving these pathological states.

| Pro-fibrotic miRNAs in diabetic complications. TGF-β increases miR-21 expression through Smad3 activation. miR-21 expression is also directly increased by TGF-β and high glucose. miR-21 can in turn activates EndMT through releasing PTEN of Smad7 inhibition (red arrow). Indeed, both PTEN and SMAD7 are negative regulators of EndMT via the Akt and TGF-β1/Smad3 signaling respectively. SMAD7 can also suppress fibrosis by down-regulating the pro-fibrotics miR-21 and miR-192, and up-regulating the anti-fibrotic miR-29b. miR451 triggers EndMT by blocking AMPK, an inhibitor of the TGF-β/SMAD pathway. miR-449a induces EndMT by inhibiting AdipoR2 and E-cadherin interaction in the lipid rafts. miR-374b plays its profibrotic activity by releasing MAPK7/ERK5-mediated EndMT inhibition. Finally, miR-122 activates EndMT via the neuronal PAS domain protein 3 (NPAS3). Pro-fibrotic miRNAs are shown in dark, anti-fibrotic miRNAs in red.

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Anti-fibrotic miRNAs in diabetic complications.

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Pro-fibrotic miRNAs in diabetic complications.

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fcell-09-683594 May 12, 2021 Time: 17:51 # 1

REVIEW

published: 19 May 2021

doi: 10.3389/fcell.2021.683594

Edited by:

Isotta Chimenti,

Sapienza University of Rome, Italy

Reviewed by:

Subrata Chakrabarti,

Western University, Canada

Swayam Prakash Srivastava,

Yale University, United States

*Correspondence:

Gheyath K. Nasrallah

gheyath.nasrallah@qu.edu.qa

Arduino Aleksander Mangoni

arduino.mangoni@ﬂinders.edu.au

Gianfranco Pintus

gpintus@sharjah.ac.ae

Specialty section:

This article was submitted to

Molecular Medicine,

a section of the journal

Frontiers in Cell and Developmental

Biology

Received: 21 March 2021

Accepted: 26 April 2021

Published: 19 May 2021

Citation:

Giordo R, Ahmed YMA, Allam H,

Abusnana S, Pappalardo L,

Nasrallah GK, Mangoni AA and

Pintus G (2021) EndMT Regulation by

Small RNAs in Diabetes-Associated

Fibrotic Conditions: Potential Link

With Oxidative Stress.

Front. Cell Dev. Biol. 9:683594.

doi: 10.3389/fcell.2021.683594

EndMT Regulation by Small RNAs in

Diabetes-Associated Fibrotic

Conditions: Potential Link With

Oxidative Stress

Roberta Giordo1, Yusra M. A. Ahmed1, Hilda Allam1, Salah Abusnana2,3 ,

Lucia Pappalardo4, Gheyath K. Nasrallah5,6*, Arduino Aleksander Mangoni7,8*and

Gianfranco Pintus1,9*

1Department of Medical Laboratory Sciences, College of Health Sciences and Sharjah Institute for Medical Research,

University of Sharjah, Sharjah, United Arab Emirates, 2Department of Diabetes and Endocrinology, University Hospital

Sharjah, Sharjah, United Arab Emirates, 3Department of Clinical Sciences, College of Medicine, University of Sharjah,

Sharjah, United Arab Emirates, 4Department of Biology, Chemistry and Environmental Studies, American University

of Sharjah, Sharjah, United Arab Emirates, 5Department of Biomedical Sciences, College of Health Sciences Member of QU

Health, Qatar University, Doha, Qatar, 6Biomedical Research Center, Qatar University, Doha, Qatar, 7Discipline of Clinical

Pharmacology, College of Medicine and Public Health, Flinders University, Adelaide, SA, Australia, 8Flinders Medical Centre,

Adelaide, SA, Australia, 9Department of Biomedical Sciences, University of Sassari, Sassari, Italy

Diabetes-associated complications, such as retinopathy, nephropathy, cardiomyopathy,

and atherosclerosis, the main consequences of long-term hyperglycemia, often lead

to organ dysfunction, disability, and increased mortality. A common denominator of

these complications is the myoﬁbroblast-driven excessive deposition of extracellular

matrix proteins. Although ﬁbroblast appears to be the primary source of myoﬁbroblasts,

other cells, including endothelial cells, can generate myoﬁbroblasts through a process

known as endothelial to mesenchymal transition (EndMT). During EndMT, endothelial

cells lose their typical phenotype to acquire mesenchymal features, characterized by

the development of invasive and migratory abilities as well as the expression of typical

mesenchymal products such as α-smooth muscle actin and type I collagen. EndMT

is involved in many chronic and ﬁbrotic diseases and appears to be regulated by

complex molecular mechanisms and different signaling pathways. Recent evidence

suggests that small RNAs, in particular microRNAs (miRNAs) and long non-coding

RNAs (lncRNAs), are crucial mediators of EndMT. Furthermore, EndMT and miRNAs are

both affected by oxidative stress, another key player in the pathophysiology of diabetic

ﬁbrotic complications. In this review, we provide an overview of the primary redox signals

underpinning the diabetic-associated ﬁbrotic process. Then, we discuss the current

knowledge on the role of small RNAs in the regulation of EndMT in diabetic retinopathy,

nephropathy, cardiomyopathy, and atherosclerosis and highlight potential links between

oxidative stress and the dyad small RNAs-EndMT in driving these pathological states.

Keywords: EndMT, miRNAs, diabetes, ﬁbrosis, oxidative stress

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Giordo et al. Small RNAs Regulate Diabetes-Associated EndMT

INTRODUCTION

Diabetes mellitus (DM) is one of the most common chronic

diseases worldwide (Lin X. et al., 2020). A prediction study

estimated a signiﬁcant further increase in the number of people

suﬀering from diabetes, especially in developing countries, with

a global prevalence of 7.7% (439 million adults) by 2030 (Shaw

et al., 2010;Lin X. et al., 2020). Long-term hyperglycemia is

the main driver of the onset and the progression of common

diabetic complications, particularly those aﬀecting the eye,

kidney, nervous system, and cardiovascular system (Deshpande

et al., 2008). Such complications are secondary to structural and

functional alterations of organs and tissues that are caused by

an increased cellular glucose uptake (Wellen and Hotamisligil,

2005). This activates inﬂammatory pathways which ultimately

leads to excessive deposition of extra cellular matrix (ECM)

proteins and consequent thickening of the vessel wall (Wellen

and Hotamisligil, 2005;Wynn, 2008). Tissue ﬁbrosis is therefore

the common denominator of most diabetic complications,

including atherosclerosis, cardiomyopathy, nephropathy and

retinopathy (Ban and Twigg, 2008). Myoﬁbroblasts are the

key mediators of pathological ECM accumulation (Kendall and

Feghali-Bostwick, 2014). These cells are normally involved in

tissue repair and are subsequently removed by apoptosis at

the end of the repair process. However, under pathological

situations, their unrestrained activation leads to excessive ECM

deposition (Micallef et al., 2012). Myoﬁbroblasts originate from

diﬀerent precursor cells, depending on the organ and the

type of initial injury (Bochaton-Piallat et al., 2016). Although

ﬁbroblasts represent the primary source of myoﬁbroblasts, the

latter can also originate from the inresident or bone marrow-

derived mesenchymal cells as well as epithelial and endothelial

cells (ECs), through a process known as epithelial/endothelial

to mesenchymal transition (Micallef et al., 2012;Kendall

and Feghali-Bostwick, 2014). In particular, endothelial to

mesenchymal transition (EndMT), the process involving ECs, is

emerging as an important player in the pathogenesis of diabetic

ﬁbrosis (Srivastava et al., 2013;Cao et al., 2014;Souilhol et al.,

2018). ECs, constituting the inner layer of blood vessels, are

responsible for maintaining vascular homeostasis in response

to endogenous and exogenous perturbations (Sandoo et al.,

2010;Khaddaj Mallat et al., 2017). There is good evidence

that ECs, when exposed to hyperglycemia, undergo signiﬁcant

alterations that result in an imbalance between vasodilation and

vasoconstriction as well as the development of inﬂammatory

and vascular complications (Bakker et al., 2009;Meza et al.,

2019). Moreover, high glucose concentrations have been shown

to trigger the shift of the endothelium toward the mesenchymal

phenotype (Yu et al., 2017;Giordo et al., 2021). Overall,

EndMT appears to represent the key link in the interaction

between inﬂammation and endothelial dysfunction in diabetic

complications (Cho et al., 2018;Man et al., 2019). In the setting

of EndMT, ECs lose their typical cobblestone morphology and

tight junctions and acquire increased motility and the ability

to secrete ECM proteins (Dejana et al., 2017). In addition,

concurrently with the loss of typical endothelial markers, such as

vascular endothelial cadherin (VE-cadherin), platelet endothelial

cell adhesion molecule (PECAM-1), also known as CD31, and

von Willebrand Factor (vWF), they acquire the ability to express

several mesenchymal markers, such as alpha-smooth muscle

actin (α-SMA), smooth muscle protein 22 alpha (SM22α),

ﬁbronectin, vimentin, and ﬁbroblast speciﬁc protein-1 (FSP-1)

(Dejana et al., 2017;Hong et al., 2018). EndMT is involved

in many chronic and ﬁbrotic disease states and appears to be

regulated by several factors (Evrard et al., 2016;Thuan et al., 2018;

Phan et al., 2020). In diabetes, oxidative stress is emerging as an

important trigger of the ECs transformation into myoﬁbroblasts

and vascular remodeling (Montorfano et al., 2014;Thuan et al.,

2018). Indeed, hyperglycemia can increase the production of

reactive oxygen species (ROS), which in turn activate signaling

pathways leading to the disruption of ECs hemostasis (Russell

et al., 2002;Peng et al., 2013;Li et al., 2017;Volpe et al.,

2018). Several signaling pathways have been demonstrated to

be involved in EndMT regulation, e.g., transforming growth

factor-beta (TGF-β) signaling, Notch signaling, ﬁbroblast growth

factor/ﬁbroblast growth factor receptor 1 (FGF/FGFR1) signaling

pathway, Smad2/3-mediated pathways (Piera-Velazquez and

Jimenez, 2019) and pro-inﬂammatory signaling cascades (Lin

et al., 2018;Ferreira et al., 2019). An important role in the

regulation of EndMT is also played by micro RNAs (miRNAs),

a class of short endogenous non-coding RNAs that regulate gene

expression at post-transcriptional level by binding to the 30-

untranslated region of messenger RNA (mRNA) (Kim et al.,

2015;Michlewski and Cáceres, 2019). A single miRNA can target

multiple mRNAs, thus inﬂuencing several processes such as cell

diﬀerentiation, proliferation, and apoptosis (Vidigal and Ventura,

2015). miRNAs can also target signiﬁcant parts of pathways

since miRNAs with similar (seed) sequence target similar sets

of genes and thus similar sets of pathways (Kehl et al., 2017).

Moreover miRNAs can, either positively or negatively, regulate

gene expression (Catalanotto et al., 2016). As a result, they

represent promising markers and druggable targets for many

diseases, including diabetes (Regazzi, 2018;Cao et al., 2019;Fan

et al., 2020). An increasing amount of evidence also suggests

that diabetes progression is linked to the alteration of miRNAs

expression proﬁles; indeed, proﬁbrotic miRNAs, such as miR-

125b, let-7c, let-7g, miR-21, miR-30b, and miR-195 have been

shown to be upregulated in EndMT. By contrast, antiﬁbrotic

miRNAs, such as miR-122a, miR-127, miR-196, and miR-375,

with inhibitory action toward genes responsible for EndMT, have

been shown to be downregulated (Ghosh et al., 2012;Kim, 2018;

Srivastava et al., 2019). In addition to miRNAs, recent studies

have also demonstrated the involvement of another class of small

RNAs, known as long non-coding RNAs (lncRNAs), in diabetes-

associated EndMT (Feng et al., 2017;Leung and Natarajan, 2018).

Compared to miRNAs, the concentrations of lncRNAs are almost

tenfold lower, with the latter exhibiting signiﬁcant tissue and cell

speciﬁcity (Cabili et al., 2011). However, the knowledge of the

function and the regulation of lncRNAs are still limited. This

review aims to summarize and discuss the available knowledge

on the role of small RNAs in the regulation of EndMT in

diabetes-associated ﬁbrotic complications such as retinopathy,

nephropathy, cardiomyopathy, atherosclerosis, and its potential

link with oxidative.

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DIABETIC NEPHROPATHY

Diabetic nephropathy (DN) is the leading cause of chronic

kidney disease in about 40% of patients with type 1 and type

2 diabetes (Gross et al., 2005). Poorly controlled blood glucose

concentrations can damage the ﬁltering functionality of the

kidneys, which become unable to remove waste products and

extra ﬂuids from the body (Rheinberger and Böger, 2014;Ruiz-

Ortega et al., 2020). The symptoms of DN do not generally

manifest in the early stages, but rather when kidney function has

signiﬁcantly deteriorated (Lim, 2014). Therefore, a tight blood

glucose control is key to prevent the onset and progression

of DN (Lewis and Maxwell, 2014;Ruiz-Ortega et al., 2020).

The progression of DN is deﬁned by various clinical stages

which reﬂect the gradual involvement of tissue damage to

diﬀerent kidney compartments: glomerulus, tubules, vasculature

and interstitium (Mogensen et al., 1983). The ﬁnal stage of DN

is characterized by renal ﬁbrosis and organ failure, which are

the result of the excessive accumulation of ECM (Calle and

Hotter, 2020). Renal ﬁbrosis is driven by multiple mechanisms,

including glucose metabolism abnormalities associated with

oxidative stress, inﬂammatory processes, and hemodynamic

changes (Brosius, 2008). Consequently, many signaling pathways

and cell types (mesangial cells, endothelial cells and podocytes)

are involved in the ﬁbrotic process (Badal and Danesh, 2014;

Aghadavoud et al., 2017). As mentioned above, alterations of

glucose metabolism not only activate various signaling pathways

(Badal and Danesh, 2014;Aghadavoud et al., 2017) but also

induce oxidative stress, a key pathophysiological step in the onset

and progression of diabetes-associated vascular complications

(Kashihara et al., 2010;Mima, 2013;Oguntibeju, 2019). Indeed,

high glucose concentrations activate the diacylglycerol-protein

kinase C (DAG-PKC) pathway, which is associated with

endothelial dysfunction, increased production of extracellular

matrix and activation of cytokines and transforming growth

factor-β(TGF-β) (Koya and King, 1998;Evcimen and King,

2007). In addition, protein kinase C (PKC) induces oxidative

stress by activating mitochondrial NADPH oxidase (Chen

et al., 2014;Giordo et al., 2021). Increased glucose can also

activate aldose reductase and the polyol pathway, leading to

the depletion of Nicotinamide Adenine Dinucleotide Phosphate

(NADPH), which is also required for the generation of the

cellular antioxidant nitric oxide (NO) (Tesfamariam, 1994;

Hummel et al., 2006;Ying, 2008;Zhao et al., 2008). The

reduced NO availability compromises the balance between

ROS generation and antioxidant defense, one of the leading

causes of endothelial dysfunction (Schiﬀrin, 2008). Furthermore,

hyperglycemia enhances the formation of advanced glycation end

products (AGEs), proteins or lipids that become glycated as a

result of exposure to sugars (Goldin et al., 2006). AGEs increase

ROS production and promote inﬂammation and ﬁbrosis through

the activation of PKC, the nuclear factor kappa light chain

enhancer of activated B cells (NF-kB) and TGF-β(Aghadavoud

et al., 2017;Rhee and Kim, 2018). Within the hemodynamic

factors driving renal ﬁbrosis, an important role is played by

the over-activation of the renin-angiotensin-aldosterone system

(RAAS), a crucial hormone system in blood pressure regulation

and ﬂuid balance (Benigni et al., 2010;Patel et al., 2017).

Hyperglycemia and insulin resistance increases the release of

angiotensin II (Ang II) a potent vasoconstrictor belonging

to the RAAS system (Giacchetti et al., 2005;Benigni et al.,

2010;Williams and Scholey, 2018). Angiotensin II plays an

important role in renal ﬁbrosis by activating a number of factors

responsible for ECM production such as TGF-β, PKC and NF-

κB (Badal and Danesh, 2014;Aghadavoud et al., 2017). On

the other hand, Angiotensin-converting enzyme2 (ACE2), the

main modulator of the RAAS system (Benigni et al., 2010),

prevents the accumulation of Ang II by catalyzing the conversion

of Ang II into the vasodilator Angiotensin I (Ang I) (Batlle

et al., 2010;Williams and Scholey, 2018). Although no cure

is available for DN, the control of blood sugar levels and

blood pressure, together with a healthy lifestyle, can slow or

stop its progression. The most common DN treatments are

based on the RAAS system inactivation; precisely with the use

of either the ACE inhibitors (ACEis) or angiotensin receptor

blockers (ARBs) or their combination (Anand and Tamura,

2012;Pathak and Dass, 2015). This type of treatments allows

the lowering of proteinuria and the blood pressure within the

glomerular capillaries. In addition, ACEis can also ameliorates

kidney ﬁbrosis in combination with other drugs. Is this the case

of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) an antiﬁbrotic

peptide that, in combination with the ACEi, imidapril, improves

kidney ﬁbrosis restoring antiﬁbrotic miRNAs, such as miR-29

and miR-let-7 and increasing the inhibition of the proﬁbrotic

dipeptidyl peptidase-4 (DPP-4) (Nitta et al., 2016;Srivastava et al.,

2020a). DPP-4 inhibitors are another class of medicines used

for DN’s treatment. In this context, due to the highest aﬃnity

for DPP-4, the drug Linagliptin is one of the most widely used

(Kanasaki, 2018). In addition, promising data also come from

treatments aiming at restoring Sirtuin 3 (SIRT3), which appear to

ameliorate renal damage, via inhibition of aberrant glycolysis and

preserving mitochondrial homeostasis (Srivastava et al., 2018;

Locatelli et al., 2020).

miRNAs REGULATION OF

DN-ASSOCIATED EndMT

The ECM is a three-dimensional network of macromolecules

(proteoglycans and ﬁbrous proteins), present in all tissues and

organs, that contributes to tissue morphogenesis, diﬀerentiation

and homeostasis. Collagens, elastins, ﬁbronectins, and laminins

are the main proteins constituting the ECM (Frantz et al., 2010;

Yue, 2014). The excessive deposition of ECM components is the

hallmark of ﬁbrosis, which represents a key pathophysiological

step in many chronic inﬂammatory diseases, including diabetes

(Herrera et al., 2018). Myoﬁbroblasts are the main cellular

mediators of ﬁbrosis as they have the ability to invade the

interstitial space and produce excessive amounts of ECM proteins

(Zent and Guo, 2018). Although resident mesenchymal cells

are the main source of myoﬁbroblasts, the latter can also

derive from other type of cells including pericytes, ﬁbrocytes,

epithelial and endothelial cells (ECs). The process involving ECs,

known as EndMT, has been shown to actively contribute to

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Giordo et al. Small RNAs Regulate Diabetes-Associated EndMT

the progression of renal ﬁbrosis (Zeisberg et al., 2008;Curci

et al., 2014;Sun et al., 2016). Besides, the mesenchymal shift

contribution to kidney ﬁbrosis can also be accelerate by the

crosstalk between endothelium and epithelium, since EndMT can

inﬂuence and induce EMT in tubular cells (Li et al., 2020b).

In this context, N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP)

plays a crucial role in inhibiting both EndMT and EndMT-

mediated EMT. Its inhibitory action is exerted by targeting

the ﬁbroblast growth factor receptor 1 (FGFR1), an antiﬁbrotic

endothelial receptor (Li et al., 2020b), and by controlling the

metabolic switch between glucose and fatty acid metabolism.

Indeed, defects in normal kidney metabolism can accelerate

EndMT and EndMT-mediated EMT contributing to kidney

ﬁbrosis (Srivastava et al., 2018, 2020b). An increasing body of

evidence suggests that miRNAs are key regulators of EndMT as

they appear diﬀerentially expressed under ﬁbrotic stimuli such

as high glucose, TGFβ, and hypoxia (Glover et al., 2019). This

diﬀerential expression also reﬂects the speciﬁc role, proﬁbrotic or

antiﬁbrotic, played by miRNAs (Hulshoﬀ et al., 2019;Srivastava

et al., 2019). The most potent inducer of kidney ﬁbrosis is

TGF-β(Wang J. et al., 2016;Wang Z. et al., 2017), which

can trigger EndMT either by activation of speciﬁc signaling

pathways, such as Akt and Smad (Wang J. et al., 2016;Wang

Z. et al., 2017), or by increasing the expression of pro-ﬁbrotic

miRNAs (Srivastava et al., 2019). In this context, TGF-βmediates

EndMT through the up-regulation of miR-21, a key modulator of

ﬁbrosis (Srivastava et al., 2013;Huang et al., 2015). Speciﬁcally,

TGF-βelicits miR-21 increase through the activation of Smad3

which regulates miR-21 expression both at a transcriptional and

a post-transcriptional level (Zhong et al., 2011). In addition,

Smad3 modulates the expression of other miRNAs and activates

the expression of various ﬁbrotic genes (Loboda et al., 2016).

Another mechanism used by miR-21 to stimulate renal ﬁbrosis

is the inhibition of Smad7 protein, a negative regulator of TGF-

β1/Smad3 signaling. In this context, Smad7 has been shown to

suppress renal ﬁbrosis by down-regulating pro-ﬁbrotic miRNAs

such as miR-21 and miR-192 while up-regulating the anti-

ﬁbrotic miR-29b (Chung et al., 2013;Loboda et al., 2016).

Additionally, miR-21 also regulates TGF-β-mediated EndMT

through the PTEN/Akt pathway (Kumarswamy et al., 2012).

Speciﬁcally, TGF-βincreases the endothelial expression of miR-

21, which in turn decreases the expression of PTEN, ultimately

promoting EndMT by Akt activation (Meadows et al., 2009;

Medici et al., 2011;Kumarswamy et al., 2012). Another molecule

linked to TGF-βsignaling in kidney ﬁbrosis is the dipeptidyl

peptidase-4 (DDP-4), a multi-functional protein expressed on the

surface of most cell types, including ECs (Deacon, 2019). DPP-

4 overexpression induces TGF-β-mediated EndMT in diabetic

nephropathy (Shi et al., 2015;Kanasaki, 2016). Furthermore,

recent studies have reported a relationship between DPP-4 and

miR-29 in diabetic kidney ﬁbrosis, where the overexpression of

DPP-4 results associated with the suppression of miR-29s family

anti-ﬁbrotic activity (Kriegel et al., 2012;Harmanci et al., 2017).

In line with these observations, the use of the DPP-4 inhibitor,

linagliptin, ameliorates kidney ﬁbrosis by restoring miR-29s and

consequentially inhibiting EndMT in diabetic mice (Kanasaki

et al., 2014). The anti-ﬁbrotic peptide, AcSDKP which suppresses

the TGF-β-induced EndMT in diabetic kidney (Nagai et al., 2014;

Hrenak et al., 2015) can also, alone or in combination with

angiotensin-converting enzyme inhibitor (ACEi), ameliorates

renal ﬁbrosis by suppressing DPP-4 and restoring the anti-

ﬁbrotic miR-29s and miR-let-7s expression in TGF-β-induced

EndMT (Srivastava et al., 2020a). The crosstalk between miR-

29s and miR-let-7s is crucial for maintaining endothelial cell

homeostasis and AcSDKP potentiates this crosstalk regulation

(Srivastava et al., 2019). Indeed, the presence of AcSDKP

upregulates the antiﬁbrotic miR-let-7 families, especially miR-

let-7b, which suppress TGFβR1 and TGFβsignaling (Srivastava

et al., 2016). Suppression of TGFβsignaling results in the up-

regulation of the miR-29 family expression, which in turn induce

FGFR1 phosphorylation, a critical step for miR-let-7 production

(Srivastava et al., 2016, 2019). The associated expression of miR-

29 and miR-let-7 is also regulated by an alternative mechanism

involving interferon-gamma (IFNγ) (Srivastava et al., 2019).

Precisely, miR-29 target the proﬁbrotic IFNγ(Ma et al., 2011)

blocking its inhibitory action toward FGFR1 which in turn

induces the expression of miR-let-7 (Chen et al., 2012;Srivastava

et al., 2019). Although not strictly related to DN, an additional

anti-ﬁbrotic mechanism, occurring by the suppression of DPP-

4, involves miR-448-3p. EndMT inhibition and amelioration of

vascular dysfunction has been indeed observed in both diabetic

mice and cell models overexpressing miR-448-3p (Guan et al.,

2020). A further regulatory mechanism of EndMT in diabetic

nephropathy involves miR-497 and its two targets, ROCK1 and

ROCK2, which belong to the rho-associated kinases (ROCKs)

family and are activated in diabetes (Kolavennu et al., 2008;

Liu et al., 2018;Matoba et al., 2020). A recent study showed

that ROCKs inhibition, following treatment with melatonin

(N-acetyl-5-methoxytryptamine), suppressed TGF-β2-induced

EndMT. Speciﬁcally, the negative modulation of ROCK1 and

ROCK2 is associated with the melatonin-induced up-regulation

of miR-497, both in glomerular cells and diabetic rats (Liu

et al., 2018). See ﬁgures and associated tables to overview of the

signaling pathways involving both anti-ﬁbrotic (Figure 1 and

Table 1) and pro-ﬁbrotic (Figure 2 and Table 2) miRNAs.

DIABETIC CARDIOMYOPATHY

Diabetic cardiomyopathy (DCM), another common

complication in diabetes, refers to myocardial dysfunction

in the absence of conventional cardiovascular complications

(coronary artery disease, valvular disease) and risk factors

(hypertension, dyslipidemia) (Boudina and Abel, 2010;Jia et al.,

2018). In the early stages, DCM is usually asymptomatic and

characterized by left ventricular (LV) hypertrophy, LV diastolic

dysfunction with diastolic ﬁlling abnormalities, myocardial

ﬁbrosis and cell signaling abnormalities. Disease progression

leads to systolic dysfunction (left ventricular low ejection

fraction) accompanied by heart failure, which is characterized

by marked hypertrophy and ﬁbrosis in the advanced stages

(Boudina and Abel, 2010;Jia et al., 2018;Tan et al., 2020).

Hyperglycemia, insulin resistance, lipid metabolism defects

and oxidative stress up-regulate the production of advanced

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Giordo et al. Small RNAs Regulate Diabetes-Associated EndMT

FIGURE 1 | Anti-ﬁbrotic miRNAs in diabetic complications. miR-142-3p and miR-200b inhibit EndMT by inactivating the TGF-β-SMAD pathway. The antiﬁbrotic

activity of miR-200b is played by down-regulating the TGF-β/SMAD pathway coactivator p300. miR-497 suppresses TGF-β-induced EndMT by ROCK1 and ROCK2

inactivation. The overexpression of DPP-4 is associated with the suppression of the miR-29s family anti-ﬁbrotic activity. However, both linagliptin and AcSDKP

suppresses EndMT by restoring miR-29 and miR-let-7s activities. Furthermore, miR-448-3p inhibits EndMT via DPP-4 suppression. AcSDKP upregulates the

antiﬁbrotic miR-let-7 which suppresses TGFβR1 and TGFβsignaling. The block of TGFβsignaling results in up-regulation of miR-29 gene expression, which in turn

causes FGFR1 phosphorylation. FGFR1 phosphorylation is critical for miR-let-7 production. miR-29 can also target the proﬁbrotic IFNY blocking its inhibitory action

toward FGFR1. The miR-29s family inhibits high glucose-induced EndMT by down-regulating Notch2, which is also suppressed by miR-18a-5p. However, DPP-4

inhibitor and AcSDKP suppresses EndMT by restoring of miR-29 and miR-let-7s activities. Furthermore, miR-448-3p inhibit EndMT via DPP-4 suppression. The

miR-29s family inhibits high glucose-induced EndMT by the downregulation of Notch2 which is also suppressed by miR-18a-5p. High glucose-induced EndMT is

also suppressed by miR-221/222 family, via the negative regulation of Wnt/β-catenin, and by miR-202-5p via inhibition of TGFβR2/TGFβsignaling pathway.

Pro-ﬁbrotic miRNAs are showed in dark, anti-ﬁbrotic miRNAs in red.

TABLE 1 | Anti-ﬁbrotic miRNAs in diabetic complications.

Anti-ﬁbrotic miRNAs in diabetic complications

miRNAs DN DR Other DCM References

miR-142-3p TGFβ-SMAD Zhu et al., 2018

miR-200b miR-200b TGFβ1-p300 TGFβ-p300 Cao et al., 2014;Feng et al., 2016

miR-202-5p TGFβR2 Gu et al., 2020

miR-497b ROCK1/2 Liu et al., 2018

miR-221/222 miR-221/222 Wnt-β/Catenin Verjans et al., 2018;Wang et al., 2020

miR-29s miR-29s TGFβsignaling Notch2 Srivastava et al., 2016, 2019, 2020a;Zhang et al., 2019

miR-Let7 TGFβsignaling Srivastava et al., 2016, 2019, 2020a

miR-448-3p TGFβsignaling Guan et al., 2020

miR-18a-5p Notch2 Geng and Guan, 2017

Summarizes the references describing the anti-ﬁbrotic miRNAs in diabetic complication.

DN, diabetic nephropathy; DR, diabetic retinopathy; DCM, diabetic cardiomyopathy.

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FIGURE 2 | Pro-ﬁbrotic miRNAs in diabetic complications. TGF-βincreases miR-21 expression through Smad3 activation. miR-21 expression is also directly

increased by TGF-βand high glucose. miR-21 can in turn activates EndMT through releasing PTEN of Smad7 inhibition (red arrow). Indeed, both PTEN and SMAD7

are negative regulators of EndMT via the Akt and TGF-β1/Smad3 signaling respectively. SMAD7 can also suppress ﬁbrosis by down-regulating the pro-ﬁbrotics

miR-21 and miR-192, and up-regulating the anti-ﬁbrotic miR-29b. miR451 triggers EndMT by blocking AMPK, an inhibitor of the TGF-β/SMAD pathway. miR-449a

induces EndMT by inhibiting AdipoR2 and E-cadherin interaction in the lipid rafts. miR-374b plays its proﬁbrotic activity by releasing MAPK7/ERK5-mediated EndMT

inhibition. Finally, miR-122 activates EndMT via the neuronal PAS domain protein 3 (NPAS3). Pro-ﬁbrotic miRNAs are shown in dark, anti-ﬁbrotic miRNAs in red.

TABLE 2 | Pro-ﬁbrotic miRNAs in diabetic complications.

Pro-ﬁbrotic miRNAs in diabetic complications

miRNAs DN DR AS DCM References

miR-21 TGFβ-SMAD Srivastava et al., 2013

miR-21 PTEN/Akt Kumarswamy et al., 2012

miR-21 NFkB/SMAD Li Q. et al., 2020

miR-451 TGFβ-SMAD Liang et al., 2019

miR-449 E-cadherin/AdipoR2 Jiang et al., 2019

miR-374b MAPK7/ERK Vanchin et al., 2019

miR-122 NPAS3 Wu et al., 2021

Summarizes the references describing the pro-ﬁbrotic miRNAs in diabetic complications.

DN, diabetic nephropathy; DR, diabetic retinopathy; DCM, diabetic cardiomyopathy; AS, atherosclerosis.

glycation end-products (AGEs) and Ang II, which in turn induce

mitochondrial dysfunction in cardiomyocytes and ECs (Tan

et al., 2002;Dikalov and Nazarewicz, 2013;Yan et al., 2014;

Brunvand et al., 2017). Mitochondrial dysfunction, as well as the

Ang II-induced NADPH oxidases stimulation, increases ROS

production and oxidative stress (Dikalov and Nazarewicz, 2013;

Siasos et al., 2018). Additionally, oxidative stress is also increased

by lipid accumulation caused by an insulin resistance-induced

cardiomyocytes metabolic shift. Indeed, the increased intake of

fatty acid is not adequately metabolized by β-oxidation resulting

in lipotoxicity (Boudina and Abel, 2010;Tan et al., 2020).

Oxidative stress can in turn trigger endoplasmic reticulum (ER)

stress, impairment of mitochondrial Ca2+uptake, cardiomyocyte

hypertrophy, ECs damage, microvascular dysfunction and the

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proﬁbrotic responses by ﬁbroblasts and inﬂammatory cells

(Boudina and Abel, 2010;Tan et al., 2020). All these eﬀects

contribute to the accumulation of ECM, especially collagen type

I and III, leading to myocardial ﬁbrosis (Jia et al., 2018;Gollmer

et al., 2019). The main signaling pathways underlying these

pathophysiological events include TGFβ/SMAD, NFκB/SMAD,

PKC, MAPK, Wnt/β-catenin, Notch2 and AcSDKP-FGFR1

signaling pathway (Nemir et al., 2014;Hu et al., 2018;Ma

et al., 2018;Hortells et al., 2019;Li et al., 2020b;Youseﬁ et al.,

2020). Most of these pathways lead to the development of

cardiac ﬁbrosis through the diﬀerentiation of ﬁbroblasts into

myoﬁbroblasts as well as the endothelial-to-mesenchymal or

epithelial-to-mesenchymal transition (Kovacic et al., 2012).

Furthermore, increasing evidence suggests that miRNAs are the

main players in the regulation of multiple pathways and cellular

processes leading to cardiac ﬁbrosis (Guo and Nair, 2017;Nandi

and Mishra, 2018;Youseﬁ et al., 2020).

miRNAs REGULATION OF

DCM-ASSOCIATED EndMT

The hyperglycemia-induced ECs damage and activation,

resulting in vascular remodeling and EndMT, has been

conﬁrmed in myocardial ﬁbrosis (Sharma et al., 2017). As

suggested by experimental evidence, cardiac ﬁbrogenesis

involves the presence of a subset of EndMT-derived activated

cardiac ﬁbroblasts (Widyantoro et al., 2010;Sharma et al.,

2017;Sánchez-Duﬀhues et al., 2018). Similarly, miRNAs are an

important regulatory mechanism in cardiac ﬁbrosis and heart

failure (Wang Z. et al., 2016;Wang and Cai, 2017). In this

context, miR-21, which has been widely described in pulmonary

and renal ﬁbrosis (Liu et al., 2016), plays an important role also

in the pathogenesis of cardiac ﬁbrosis and DCM (Adam et al.,

2012;Guo and Nair, 2017;Yuan et al., 2017;Dai et al., 2018).

A recent in vivo study conﬁrmed the involvement of miR-21

in EndMT activation and myocardial ﬁbrosis, showing that the

hyperglycemia-induced up-regulation of miR-21 in diabetic mice

is associated with the down-regulation of endothelial markers

and the up-regulation of ﬁbroblast markers (Li Q. et al., 2020).

Moreover, similarly to the mechanism described in diabetic

nephropathy (Zhong et al., 2011), miR-21 regulates EndMT

through the NF-κB-SMAD signaling pathway by targeting

SMAD7. The consequent SMAD7 inhibition increases SMAD2

and SMAD3 phosphorylation, resulting in EndMT activation (Li

Q. et al., 2020). An additional mechanism, requiring the TGF-

β/SMAD pathway, involves miR-142-3p, which has been shown

to attenuate the hyperglycemia-induced EndMT in human aortic

endothelial cells (HAECs) (Zhu et al., 2018). Indeed, miR-142-3p

overexpression inhibits EndMT by inactivating both TGF-β1

and the downstream target gene SMAD2. By contrast, TGF-β1

overexpression signiﬁcantly abolishes the inhibitory eﬀects of

miR-142-3p (Zhu et al., 2018). A negative regulation of glucose-

induced EndMT in the heart is also played by miR-200b (Feng

et al., 2016). In a recent study, the expression of speciﬁc ﬁbrotic

markers, such as vascular endothelial growth factor (VEGF)

(Yang et al., 2014), zinc ﬁnger E-box–binding homeobox (Zeb2)

(Jahan et al., 2018), and TGF-β1 (Biernacka et al., 2011) was

prevented in diabetic mice overexpressing miR-200b (Feng et al.,

2016). Moreover, miR-200b overexpression also induces the

down-regulation of p300, a transcription coactivator known to

contribute to cardiac ﬁbrosis and hypertrophy via TGF-β/SMAD

(Bugyei-Twum et al., 2014;Feng et al., 2016). Although the

inhibitory role of the whole miR-200 family is well established,

both in EMT (Korpal and Kang, 2008;Korpal et al., 2008) and

EndMT (Feng et al., 2016;Zhang et al., 2017), unexpectedly a

recent study shown that miR-200c-3p exerted the opposite eﬀect,

being able to promote EndMT and aortic graft remodeling both

in vivo and in vitro (Chen et al., 2021). Finally, a further TGF-

β/SMAD pathway-mediated regulatory mechanism involves

miR-451 whose eﬀects on EndMT are AMPK-dependent.

Indeed, miR451 knockdown in diabetic mouse hearts suppresses

EndMT through the activation of AMPK, which in turn inhibits

the TGF-β/SMAD pathway (Liang et al., 2019). As previously

mentioned, in addition to TGF-β/SMAD, other pathways

underlie the pathophysiological events leading to cardiac ﬁbrosis.

One of them is the Wnt signaling pathway, known to promote

ﬁbroblast activation and proliferation (Tao et al., 2016). On

the other hand, the anti-ﬁbrotic role of miRNA-221/222 family

has been conﬁrmed, as their down-regulation was associated

with heart failure (Verjans et al., 2018). The interplay between

Wnt and miR-222 in EndMT regulation has been recently

suggested (Wang et al., 2020); speciﬁcally, miR-222 is able

to suppress the hyperglycemia-induced EndMT and inhibit

cardiac ﬁbrosis by negatively regulating the Wnt/β-catenin

pathway in diabetic mice (Wang et al., 2020). Lastly, a further

protective eﬀect versus EndMT is exerted through the notch

pathway and involves miR-18a-5p (Geng and Guan, 2017). The

role of the notch pathway in heart development and control

of the balance between ﬁbrotic and regenerative repair in the

adult heart has been widely conﬁrmed (Nemir et al., 2014).

Moreover, Notch2 activation results essential for driving ECs

diﬀerentiation (Noseda et al., 2004;Kovacic et al., 2019) in

cardiovascular disease and for promoting EndMT independently

or in association with TGF-β/SMAD3 signaling (Fu et al., 2009;

Chang et al., 2011). Notch2 is a target of miR-18a-5p which

recently conﬁrmed its antiﬁbrotic role via the suppression of

Notch2 and consequent inhibition of hyperglycemia-induced

EndMT in human aortic valvular endothelial cells (HAVECs)

(Geng and Guan, 2017). See ﬁgures and associated tables

to overview of the signaling pathways involving both anti-

ﬁbrotic (Figure 1 and Table 1) and pro-ﬁbrotic (Figure 2 and

Table 2) miRNAs.

DIABETIC RETINOPATHY

Diabetic retinopathy (DR) is a common and severe microvascular

complication of the eye that represents the leading cause

of blindness in diabetes (Sabanayagam et al., 2016). The

prevalence increases with disease progression and consequently

with the exposure to the major risk factors, hyperglycemia

and hypertension (Ding and Wong, 2012;Lee et al., 2015).

Generally, a tight blood glucose control is cornerstone to reduce

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the risk of DR progression (Cheung and Wong, 2008). The

condition is initially characterized by an asymptomatic stage,

non-proliferative diabetic retinopathy (NPDR), that involves

increased vascular permeability and capillary occlusion. Retinal

neovascularization, by contrast, predominates in a later stage,

proliferative diabetic retinopathy (PDR) (Lechner et al., 2017;

Kusuhara et al., 2018), as consequence of hypoxia. However, as

new vessels are relatively fragile, they tend to bleed into the

macular region causing vision diﬃculties and, in the worst-case

scenario, diabetic macular edema (DME), the main cause of

blindness in DR (Wang and Lo, 2018). DME is described as

a swelling of the macula due to ﬂuid accumulation following

breakdown of the blood-retinal barrier (BRB). This event can

occur both in the PDR and in the NPDR stage (Das et al.,

2015;Romero-Aroca et al., 2016). The BRB is composed of two

distinct barriers: the outer BRB, consisting of retinal pigment

epithelium and the inner BRB, composed of endothelial cells

regulating the transport across retinal capillaries. Besides, the

BRB is established by tight cellular junctions, both in the

inner and outer barrier, as well as by the scarcity of endocytic

vesicles within cells, which further ensure the integrity of the

BRB (Klaassen et al., 2013;Díaz-Coránguez et al., 2017). In

addition, pericytes, specialized mural cells with a central role in

angiogenesis, regulate and stabilize this tight structure through

the Angiopoietin-1/Tie-2, platelet-derived growth factor (PDGF)

and TGF-βsignaling pathways (Caporarello et al., 2019;Trost

et al., 2019). BRB breakdown is a complex process involving

diﬀerent mechanisms; it can occur either in the inner BRB, the

outer BRB, or both sites. The loss of integrity of the endothelial

cell-cell junctions, the loss of pericytes and the thickening of

the basement membrane are the major alterations observed

in the inner BRB (Hammes et al., 2011;Das et al., 2015).

Several studies have shown that hyperglycemia represents the

main risk factor contributing to the pathogenesis of diabetic

retinopathy (Engerman and Kern, 1986;Das et al., 2015;

Eshaq et al., 2017). Furthermore, using a BRB model formed

by retinal pericytes, astrocytes and endothelial cells, it has

been recently reported that high glucose exposure elicits BRB

breakdown, enhances BRB permeability and reduces the levels

of junction proteins such as ZO-1 and VE-cadherin (Fresta

et al., 2020). Besides, elevated ROS as well as pro-inﬂammatory

mediators (IL-1β, IL-6) and oxidative stress-related enzymes

(iNOS, Nox2) have also been shown to be increased (Fresta

et al., 2020). The major biochemical pathways involved in the

BRB breakdown are the polyol pathway, the AGEs pathway,

the PKC pathway and the hexosamine pathway. Oxidative

stress and inﬂammation are responsible for the upregulation of

growth factors and cytokines, such as VEGF, tumor necrosis

factor (TNF), interleukins (ILs), and matrix metalloproteinases

(MMPs), which contribute to the BRB breakdown and to the

development of DME (Aiello et al., 1994;Brownlee, 2005;

Gupta et al., 2013;Das et al., 2015). Studies have conﬁrmed

the role of the pro-angiogenic factor VEGF as main modulator

of PDR and DME. VEGF is secreted by retinal pigmented

epithelial cells, pericytes, and endothelial cells in response to

hypoxia conditions caused by the obstruction and loss of retinal

capillaries (Gupta et al., 2013;Romero-Aroca et al., 2016). VEGF,

in addition to promoting neovascularization in PDR, participates

in the breakdown of the BRB via increasing permeability

of retinal vessels (Ray et al., 2004). Indeed, high levels of

VEGF increase the expression of the inﬂammatory intercellular

adhesion molecule-1 (ICAM-1) which in turn facilitates the

adhesion of leukocytes to the diabetic retinal vasculature,

promoting capillary occlusion (Aiello et al., 1994;Joussen et al.,

2002;Romero-Aroca et al., 2016).

miRNAs REGULATION OF

DR-ASSOCIATED EndMT

Hyperglycemia-induced increased production of ECM and

thickening of the vascular basement membrane is the hallmark of

diabetic retinopathy (Roy et al., 2015). As previously mentioned,

hyperglycemia promotes ﬁbrosis progression through the

generation of ECs-derived myoﬁbroblasts, EndMT. This process

has been shown to play an important role also in the pathogenesis

of DR (Cao et al., 2014). Similar to other diabetic complications,

TGF-βis an important EndMT mediator, mainly through the

activation of the SMAD signaling pathways (Van Geest et al.,

2010;Cao et al., 2014;Pardali et al., 2017). Moreover, the

transcriptional activator p300, already known for increasing the

expression of ECM proteins (Kaur et al., 2006), and miR-200b

have been described as key regulators of the TGF-β-mediated

EndMT in diabetic mice (Cao et al., 2014). Although the

speciﬁc mechanism played by miR-200b and p300 remains

partially unknown, the anti-ﬁbrotic activity of miR-200b, already

described in other diabetic complications (McArthur et al., 2011;

Feng et al., 2016), has also been conﬁrmed in DR. Speciﬁcally,

the EndMT observed in the retinas of wild-type diabetic mice

was suppressed by the overexpression of miR-200b (Cao et al.,

2014). As mentioned before, the outer BRB is composed of

tight junctions of retina pigment epithelial cells (RPECs) which

secrete various factors, nutrients and signaling molecules that

inﬂuence the surrounding tissues (Campbell and Humphries,

2013;Liu and Liu, 2019). Chronic hyperglycemia alters RPECs

functions contributing to the ﬂuid accumulation in DME and

the development of DR (Desjardins et al., 2016). Under stress

conditions RPECs cells can release large amounts of exosomes,

nanoscale vesicles that mediate many intercellular activities such

as cell-to-cell communication, immune regulation, inﬂammatory

response, extracellular matrix turnover and neovascularization

(Klingeborn et al., 2017;Liu et al., 2020). A recent study

conﬁrmed the importance of the crosstalk between ECs and

RPECs cells in the progression of ﬁbrosis in patients with DR

(Gu et al., 2020). Speciﬁcally, it was observed that hyperglycemia

increased the ability of RPECs to release miR-202-5p-enriched

exosomes. On the other hand, hyperglycemia induced EndMT

through the TGFβsignaling pathway activation in ECs. However,

when ECs were treated with RPECs-derived exosomes, the

hyperglycemia-induced TGFβsignaling pathway activation was

signiﬁcantly counteracted as well as the increased proliferation

and migration (Gu et al., 2020). In addition, miR-202-5p,

by targeting speciﬁcally TGFβR2, was responsible for the

TGFβsignaling pathway inactivation and EndMT suppression

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(Gu et al., 2020). This study, in addition to providing additional

evidence that hyperglycemia-induced EndMT involves the

activation of TGFβsignaling, also showed that the release of

miR-202-5p-enriched exosomes from RPE cells leads to the

suppression of EndMT. The RPE cells-derived exosomes are

therefore important mediators of the ECs-RPE cells crosstalk in

the development of DR (Gu et al., 2020). Additional miRNAs

involved in EndMT regulation in DR include two members of

the mi-RNA29 family, miR-29a and miR-29b, already described

in ﬁbrosis development associated with diabetic complications

(He et al., 2013;Kanasaki et al., 2014;Zhang Y. et al., 2014;

Srivastava et al., 2020a). The anti-ﬁbrotic activity of miR-

29a/b has been recently conﬁrmed also in DR where their

overexpression suppressed the hyperglycemia-induced EndMT

in human retinal microvascular endothelial cells (HRMECs)

(Zhang et al., 2019). The inhibitory eﬀect of miR-29a/b was

exerted through the down-regulation of the transmembrane

protein Notch2, known to activate morphological and functional

changes of ECs as well as promote EndMT (Tian et al.,

2017;Zhang et al., 2019). See ﬁgures and associated tables

to overview of the signaling pathways involving both anti-

ﬁbrotic (Figure 1 and Table 1) and pro-ﬁbrotic (Figure 2 and

Table 2) miRNAs.

ATHEROSCLEROSIS

Atherosclerosis (AS) is characterized by plaque formation,

secondary to the deposition of fats, cholesterol, and calcium,

which lead to ischemia and its clinical manifestations, such as

myocardial infarction and stroke (Lnsis, 2000). Although AS

is classically associated with alterations of lipid metabolism

and hypercholesterolemia (Wang H.H. et al., 2017), its

pathogenesis is more complex and involves various factors.

Endothelial dysfunction and inﬂammation are key steps in

the sequence of events leading to AS (Davignon and Ganz,

2004;Hansson, 2009). The presence of mechanical stress,

such as blood ﬂow turbulence, can activate the endothelium,

which responds by recruiting monocytes, adhesion molecules

and pro-inﬂammatory cytokines. Monocytes, facilitated by

adhesion molecules and cytokines, inﬁltrate the intima and

can diﬀerentiate in macrophages which actively participate in

lipid uptake through phagocytosis (Ilhan and Kalkanli, 2015).

Diabetes and AS share several pathological mechanisms (La Sala

et al., 2019b); indeed, the metabolic alterations that drive the

development of diabetes are also involved in the pathogenesis

of atherosclerosis (Federici and Lauro, 2005;Poznyak et al.,

2020). In addition, both type 1 and type 2 diabetes can either

induce atherosclerosis and accelerate its progression (Poznyak

et al., 2020). In this context, a crucial role is played by the

prolonged exposure to hyperglycemia and insulin resistance

which are responsible for the increased atherosclerosis-related

inﬂammation of the arterial wall (Reddy et al., 2010;Katakami,

2017). In addition to triggering the onset and progression

of diabetes, insulin resistance also promotes dyslipidemia,

hypertension and other metabolic abnormalities, important

components of the pro-atherogenic milieu (Semenkovich, 2006;

Katakami, 2017). At the same time, an insuﬃcient insulin

signaling elicits an abnormal lipid metabolism and glucose

transport and increase the production of glucose in the liver.

Pancreatic βcells respond to hyperglycemia by increasing

insulin secretion; however, the continued stimulation of β

cells leads to their progressive functional failure and diabetes

development (Cavaghan et al., 2000;Mangiaﬁco et al., 2011).

Prolonged exposure to hyperglycemia increases oxidative stress

(Yu et al., 2011;Volpe et al., 2018), the primary activator of

signaling pathways driving AS and diabetes progression (Vanessa

Fiorentino et al., 2013;Yuan et al., 2019). Overproduction

of ROS increases the formation of AGEs, modiﬁcations of

proteins or lipids that become non-enzymatically glycated

(Moreno-Viedma et al., 2016;Katakami, 2017). AGEs are

involved in each step of atherosclerosis, being responsible for

monocyte migration into the sub-endothelial space, release of

cytokines by macrophages and stimulation of vasoconstriction

(Katakami, 2017). Moreover, the binding of AGEs to the

receptor RAGE activates TGF-β, ERK, JNK, p38, NF-kB, PKC

and the polyol pathways as well as maintaining the chronic

pro-inﬂammatory state of the arterial wall (Katakami, 2017;

Yamagishi and Matsui, 2018).

miRNAs REGULATION OF

AS-ASSOCIATED EndMT

As previously mentioned, endothelial dysfunction driven by

oxidative stress plays a critical role in the development of AS.

Persistent activation of ECs induces EndMT, which contributes

to both the initiation and the progression of atherosclerosis

(Chen et al., 2015;Evrard et al., 2017). Moreover, the extent of

EndMT in the human plaque appears to be strongly correlated

with the severity of the disease (Souilhol et al., 2018). A recent

study showed the up-regulation of 17 miRNAs in atherosclerotic

plaques; among them, miR-449a, already known for its role in

lipid and cholesterol anabolism as well as inﬂammation (Zhang

H. et al., 2014), was signiﬁcantly higher compared with normal

arteries (Jiang et al., 2019). The authors reported that miR-

449a induces EndMT and promotes the development of AS

by targeting the interaction between adiponectin receptor 2

(AdipoR2) and E-cadherin in lipid rafts (Jiang et al., 2019). In

this context, miR-449a has displayed a multilevel and complex

regulatory mechanism by promoting proliferation and enhancing

the migrating ability of ECs as well as their expression of

atherosclerotic markers (Jiang et al., 2019). The ability to

induce EndMT was conﬁrmed by the reduced E-cadherin

expression concurrently with the increased expression of α-SMA

and SMAD3 (Jiang et al., 2019). miR-449a pro-atherosclerotic

properties are exerted by inhibition AdipoR2 and E-cadherin

migration into the lipid raft fractions of ECs and consequent

suppression E-cadherin-AdipoR2 of interaction. Additionally,

the authors reported that blocking miR-449a protects diabetic

mice from developing AS (Jiang et al., 2019). Similarly to

miR-449a, miR-374b was reported to be up-regulated both in

atheroprone regions from mice and pigs and in TGF-β1-treated

ECs (Vanchin et al., 2019). Additionally, the overexpression

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of miR-374b was associated with a reduction in endothelial

markers (VE-Cadherin and eNOS), and a concomitant increase

of mesenchymal markers (TAGLN and Calponin). Besides, miR-

374b was able to induce EndMT through the silencing of

the Mitogen-Activated Protein Kinase 7 (MAPK7) also known

as ERK5 (Vanchin et al., 2019). MAPK7 is an antagonist of

EndMT and its signaling activity is generally lost in vessel areas

that are undergoing pathological remodeling (Nithianandarajah-

Jones et al., 2014;Krenning et al., 2016). Similarly, MAPK7

signaling activity was lost in the sites of vascular remodeling,

providing an additional conﬁrmation of the inhibitory action

of miR-374b. By contrast, the recovery of MAPK7 signaling

abrogated the pathological eﬀect of miR-374b (Vanchin et al.,

2019). miR-122, another miRNA recently reported as EndMT

mediator in AS, has been shown to be up-regulated both in

the aortic intima of diabetic mice and in the cellular EndMT

model (Wu et al., 2021). The regulatory action of miR-122

is mediated by the neuronal PAS domain protein 3 (NPAS3).

Indeed, inhibition of miR-122 prevented atherosclerosis and

regulated NPAS3-mediated EndMT (Wu et al., 2021). miR-

122 might therefore represent a druggable target in preventing

EndMT-associated atherosclerosis. See ﬁgures and associated

tables to overview of the signaling pathways involving both anti-

ﬁbrotic (Figure 1 and Table 1) and pro-ﬁbrotic (Figure 2 and

Table 2) miRNAs.

LONG NON-CODING RNAs REGULATION

IN DIABETES-ASSOCIATED EndMT

Besides miRNAs, small RNAs also include long non-coding RNAs

(lncRNAs) and circular RNAs (circRNAs) which are emerging as

key regulators implicated in a signiﬁcant number of biological

processes (Qu et al., 2017;Statello et al., 2020). Unlike linear

RNAs, circRNAs form a covalently closed continuous loop,

without 50or 30ends (Qu et al., 2015). lncRNAs are instead

linear RNAs, with a nucleotide length >200, that can aﬀect

gene transcription both at the epigenetic, transcriptional and

post-transcriptional level (Dykes and Emanueli, 2017;Wang

C. et al., 2017). Thus, lncRNAs can diﬀerently interact with

mRNAs, proteins, and DNA elements; moreover, the binding

of transcriptional factors to the lncRNA promoter’s target sites

can regulate their expression (Taft et al., 2010). lncRNAs are

also precursors of many types of miRNAs, although more

frequently they overlap both physically and functionally with

the latter. Moreover, lncRNAs compete with miRNAs for the

binding to the same target genes and can trigger miRNAs

degradation (Taft et al., 2010;Chen et al., 2018). Hence,

lncRNAs are involved in a variety of human diseases where

they appear diﬀerentially expressed or genetically perturbed

(Harries, 2012;Shi et al., 2013). In this context, most of

the knowledge pertaining to lncRNAs is derived from cancer

however there is increasing evidence of their involvement in

other conditions, such as Alzheimer’s disease, diabetes, cardiac

complications (DiStefano, 2018;Greco et al., 2018;Leung and

Natarajan, 2018) and ﬁbrosis (He Z. et al., 2020;Li et al., 2020a;

Lin J. et al., 2020). One important function of lncRNAs is

their role as a molecular sponge to certain miRNAs, hindering

their expression (Biswas et al., 2018). This mechanism has been

conﬁrmed in diabetic kidney ﬁbrosis, where the down-regulation

of the anti-ﬁbrotic miR-29 was associated with lncRNA H19

up-regulation, whereas its knockdown restored miR-29 activity

and signiﬁcantly inhibited TGF-β2-induced EndMT in diabetic

mice (Shi et al., 2020). However, the role of H19 in diabetes-

associated EndMT remains unclear; indeed, H19 overexpression

prevented glucose-induced EndMT by reducing the TGF-β1

levels in DR (Thomas et al., 2019). Further studies are required

to clarify the role of H19 in regulating EndMT in diabetic

conditions. Another lncRNA involved in DR is the maternally

expressed gene 3 (MEG3) which showed an inhibitory eﬀect

on hyperglycemia-induced EndMT. MEG3 resulted indeed able

to suppress EndMT both in vivo and in vitro by inhibiting

the PI3K/AKT/mTOR signaling pathway (He Y. et al., 2020).

On the other hand, MEG3 methylation mediated by DNA

methyltransferase 1 (DNMT1) attenuated MEG3 expression and

consequently accelerated EndMT (He Y. et al., 2020). This

ﬁnding clariﬁes the role of MEG3 in EndMT and provide

additional conﬁrmation that increased levels of DNA methylation

represent a potential risk factor for the development of DR

(Maghbooli et al., 2015). As previously reported, oxidized low

density lipoproteins (ox-LDL), being able to trigger plaque

formation and EndMT, are key players in AS development

(Su et al., 2018). A recent study reported that miR-30c-5p

and LINC00657, also known as non-coding RNA activated

by DNA damage (NORAD), are both involved in ox-LDL-

induced EndMT but with opposite eﬀects (Wu et al., 2020).

miR-30c-5p inhibited ox-LDL-induced EndMT via activation of

the Wnt7b/β-catenin pathway whereas LINC00657, acting as

sponge of miR-30c-5p, suppressed the EndMT inhibition (Wu

et al., 2020). Indeed, the expression level of LINC00657 resulted

elevated both in sera from AS patients and in ox-LDL-stimulated

ECs (Wu et al., 2020).

POTENTIAL ROS-EndMT-SMALL RNAs

INTERPLAY IN DIABETES-ASSOCIATED

FIBROTIC CONDITIONS

Oxidative stress is a key player in the diabetic complications’

pathophysiology described in this review. Hyperglycemia is

not only the main factor responsible for the increase in

ROS but also favors the increase of inﬂammatory mediators,

which ultimately leads to vascular dysfunction (Luc et al.,

2019). Both genetic and epigenetic factors can regulate the

development and exacerbation of oxidative stress; in this

context, diﬀerent studies have highlighted the key role played

by miRNAs (Grieco et al., 2019). Indeed, hyperglycemia can

alter miRNAs expression, which in turn contributes to the

development of endothelium dysfunction and diabetic vascular

disease (Luc et al., 2019). Besides, in diabetic complications

the molecular mechanisms and signaling pathways triggered by

oxidative stress appear similar to those involved in miRNAs

regulation (Grieco et al., 2019;Qadir et al., 2019). Finally,

hyperglycemia-induced oxidative stress can aﬀect the expression

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Giordo et al. Small RNAs Regulate Diabetes-Associated EndMT

of speciﬁc miRNAs, which in turn can exacerbate oxidative

stress, in addition to regulating the ﬁbrotic process through

the mechanisms summarized in this review (Grieco et al.,

2019;Qadir et al., 2019). On the other hand, oxidative

stress is emerging as a key trigger of EndMT (Montorfano

et al., 2014;Thuan et al., 2018). Therefore, although a

direct oxidative stress-small RNAs-EndMT link has not been

demonstrated in diabetes yet, a substantial body of evidence

supports this interplay. For example, an indirect proof of a

ROS-miR-21-EndMT link has been reported with kallistatin, an

endogenous protein with beneﬁcial eﬀects on EndMT-associated

ﬁbrosis (Guo et al., 2015). Kallistatin treatment blocked TGF-

β-induced EndMT, NADPH oxidase-dependent ROS formation

and the expression of the pro-ﬁbrotic miR-21, conﬁrming

the role of both miR-21 and ROS as major mediators of

EndMT (Guo et al., 2015). Many studies indicated a direct

link between mi-R21 and oxidative stress in diabetic subjects,

where ROS generation has been suggested as a downstream

eﬀect of miR-21 overexpression (La Sala et al., 2019a). The pro-

oxidant eﬀect of miR-21 is exerted through the suppression

of genes which usually limit oxidative damage such as KRIT1

(Krev/Rap1 Interaction Trapped-1), Nuclear Factor erythroid

Related Factor 2 (NRF2), and MnSOD2 (Manganese-dependent

Superoxide Dismutase2). By contrast, inhibition of miR-21

decreases ROS levels (La Sala et al., 2018;Grieco et al., 2019).

A relationship between up-regulation of miR-21 and increased

ROS levels has also been shown during the development

of diabetic cardiac dysfunctions (Yildirim et al., 2013). The

miR-200 family, the anti-ﬁbrotic activity of which has been

described both in diabetic nephropathy and retinopathy, has

also been shown to be associated with a decrease in oxidative

stress in diabetes; speciﬁcally, the antioxidant eﬀect of miR-

200 is exerted by silencing the O-GlcNAc transferase, also

known as OGT, whose enzymatic activity is associated with

diabetic complications and endothelial inﬂammation (Qadir

et al., 2019). Another proof of the oxidative stress-small RNAs-

EndMT interconnection comes from a study investigating

the activity of miR-451 (Ruknarong et al., 2021). The latter,

previously described for its ability to induce EndMT in diabetic

mouse heart (Liang et al., 2019), has been recently reported

to be up-regulated in diabetic subjects with high oxidative

stress. The association between miR-451 and oxidative stress

has been further conﬁrmed with the use of the antioxidant

Vitamin C; indeed, Vitamin C administration in diabetic

subjects decreased both the expression of miR-451 and ROS

levels (Ruknarong et al., 2021). Finally, an interplay being the

basis of mitochondrial functions in kidney ECs involves the

miR-let-7 family, (FGF)/FGFR1 signaling pathway and SIRT3

(Srivastava et al., 2020c). The integrity of the FGFR1-miR-let-

7 axis, on which depends the modulation of SIRT3, is crucial

for maintaining the mitochondrial functionality (Srivastava

et al., 2020c). SIRT3, for its part, controls mitochondrial redox

homeostasis by modulation of ROS levels (Jing et al., 2011;

Bause and Haigis, 2013) mainly via activation of the antioxidant

enzyme superoxide-dismutase 2 (Qiu et al., 2010). On the

contrary, the loss of the FGFR1-miR-let-7axis impairs SIRT3

and miR-29 levels with consequent disruption of mitochondrial

integrity and activation of pro-mesenchymal signaling (Wnt

signaling, BMP, Notch, TGF-βsignaling) promoting EndMT

(Srivastava et al., 2020c).

CONCLUSION AND FUTURE

DIRECTIONS

This review has highlighted the key role of EndMT in the ﬁbrotic

process occurring in the development of the major diabetic

complications. Environmental factors (high glucose, hypoxia,

oxidative stress, pro-inﬂammatory cytokines) are important

determinants of EndMT induction through the activation

of speciﬁc signaling pathways, such as TGF-β, Notch, Wnt,

and the modulation of the expression of microRNAs. The

evidence reviewed in this article indicates that some microRNAs,

e.g., miR-29, miR-200, and miR-Let7, have anti-ﬁbrotic eﬀects

and inhibit EndMT whereas others, e.g., miR-21 and miR-

122, possess pro-ﬁbrotic properties and promote EndMT. The

anti-ﬁbrotic activity of some microRNAs appears univocal

not only within diabetic complications but also in other

pathological conditions. For instance, miR-29a/b and miR-200b

have been shown to inhibit ﬁbrosis in pulmonary ﬁbrosis

(Yang et al., 2012;Cushing et al., 2015), systemic sclerosis

(Harmanci et al., 2017) as well as in DCM, DN, and DR

(Cao et al., 2014;Kanasaki et al., 2014;Feng et al., 2016;

Zhang et al., 2019). Similarly, miR-21 is generally up-regulated

in diﬀerent ﬁbrotic diseases (Huang et al., 2015;Liu et al.,

2016) as well as in diabetic complications such as DN, DR,

and DCM (Srivastava et al., 2013;Chen et al., 2017;Li Q.

et al., 2020). Moreover, since the expression levels of miR-

21 in the plasma of diabetic patients were correlated with

disease progression, miR-21 might be used as a marker of

diabetes severity (Jiang et al., 2017). On the other hand, the

function of other microRNAs is only partially established in

in vitro models or in speciﬁc pathological conditions. Further,

for some miRNAs the evidence is still controversial, such

as the case of the lncRNA H19 which showed pro-ﬁbrotic

activity in DN (Shi et al., 2020) and an opposite eﬀect in

DR (Thomas et al., 2019). Additionally, since the markers for

EndMT used in individual studies are often diﬀerent, a complete

understanding of the regulatory mechanisms played by miRNAs,

or an exact comparison between them, is currently challenging.

In this regard, future directions in the study of diabetic

complications should involve (a) a thorough characterization

of the mechanisms involved in the ROS-EndMT-small RNAs

interplay and its relationship with the onset and severity

of speciﬁc complications, (b) the conduct of epidemiological

studies investigating the association between speciﬁc miRNAs

and lncRNAs and metabolic control, surrogate markers of

organ damage, and morbidity and mortality in patients with

diabetes, and (c) the eﬀects of speciﬁc pharmacological and

non-pharmacological interventions targeting EndMT on the risk

and progression of diabetic complications. Such studies might

contribute to the identiﬁcation of new diagnostic and therapeutic

strategies to prevent or limit the structural and functional

damage that leads to organ and system failure in diabetes.

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Giordo et al. Small RNAs Regulate Diabetes-Associated EndMT

AUTHOR CONTRIBUTIONS

RG, YMAA, and GP: conceptualization. GKN, AAM, and GP:

resources. RG and YMAA: writing the original manuscript draft.

RG, YMAA, HA, SA, LP, GKN, AAM, and GP: review and editing

the diﬀerent manuscript versions. AAM and GP: ﬁnal editing and

supervision. GP: submission. All authors: read and agreed to the

published version of the manuscript.

FUNDING

This work has been made possible thanks to grants

from the University of Sharjah (Seed 2001050151)

to GP; (collaborative 2101050160) to GP and AAM;

Qatar University (IRCC-2019-007) to GKN and GP;

and (fondo UNISS di Ateneo per la Ricerca 2020)

to GP.

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Frontiers in Cell and Developmental Biology | www.frontiersin.org 16 May 2021 | Volume 9 | Article 683594

EndMT Regulation by Small RNAs in Diabetes-Associated Fibrotic Conditions: Potential Link With Oxidative Stress

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