**2.2. Inflammation**

Endothelial dysfunction involves reduced endothelium-dependent vasodilatation and a prothrombotic, pro-inflammatory and oxidant milieu [2]. The endothelial nitric oxide (NO) synthase (eNOS), renin-angiotensin-aldosterone and kallikrein-kinin response systems all fail to maintain normal vascular homeostasis in conditions of hyperglycemia, reactive oxidative spe-

The aim of this review is to address the role of inflammation and its mechanisms in endothelial dysfunction associated with diabetes, describing the impact of these conditions on vascular function. We searched PubMed and Google Scholar primarily for original research articles published up to 2017 that were focused on the pathophysiology of endothelial dysfunction associated with type 2 diabetes. The main search terms used were "type 2 diabetes," "inflammation and endothelial dysfunction," "insulin resistance," and "therapies". We identified primarily full-text manuscripts written in English. We also searched Clinicaltrials.gov for information on

Vascular endothelium is crucial for the regulation of vascular homeostasis. It is metabolically active through the secretion of vasodilators and vasoconstrictors and acts as an active signal transducer for circulating factors that modify the vessel wall phenotype. The normal paracrine and autocrine functions of endothelial cells include the synthesis of a series of substances that moderate vascular tone, decrease leucocyte migration, control permeability, regulate proliferation and migration of smooth muscle cells, and regulate platelet adhesion and aggregation (**Figure 1**). Endothelium also regulates cellular adhesion, vessel wall inflam-

The mechanisms implicated in the genesis of endothelial dysfunction are of extreme importance in developing adequate strategies to prevent or retard the clinical manifestations of

Dysfunction of vascular endothelium is considered not only as an important factor in the initiation of vascular complications, but also in its progression and clinical sequelae [5]. Endothelial dysfunction is the loss of endothelium physiological properties with a shift toward a vasocon-

The mechanisms underlying the development of endothelial dysfunction in type 2 diabetes are complex and include oxidative stress, inflammation, and chronic alterations in the hemodynamic balance. Several contributors to endothelial activation and dysregulation have

increased arginase, increased ROS production, decreased NO bioavailability, increased asymmetric dimethyl arginine, increased glycation and expression of receptor for advanced glycation end products (RAGE), nuclear factor κB (NFκB) activation, suppression of Kruppel-like Factor 2 [6], and phenotypic changes in perivascular adipose tissue leading to low grade

) bioavailability and eNOS uncoupling,

cies (ROS), free fatty acid (FFA) stress, and pro-inflammatory signaling [3, 4].

ongoing clinical trials in endothelial dysfunction associated with type 2 diabetes.

**2. Endothelial cell function**

232 Endothelial Dysfunction - Old Concepts and New Challenges

mation, and angiogenesis.

cardiovascular diseases.

**2.1. Endothelial dysfunction in diabetes**

strictor, prothrombotic, and pro-inflammatory state [2].

been described: decreased tetrahydrobiopterin (BH<sup>4</sup>

inflammation and reduced adiponectin secretion [7, 8].

A state of subclinical systemic inflammation is characteristically present in obesity/insulin resistance and type 2 diabetes. The inflammation can be monitored by inflammatory markers such as high sensitivity C-reactive protein (hsCRP) and the inflammatory score derived from the pro-inflammatory plasma cytokines, interleukin (IL)-6, tumor necrosis factor α (TNFα), osteopontin, fractalkine, chemokine (C-C motif) ligand 2 (CCL2) and anti-inflammatory adiponectin, that inversely relate to insulin sensitivity (**Table 1**). The inflammatory score independently predicted fasting plasma glucose and insulin resistance in type 2 diabetic patients with high sensibility and specificity [9–12]. Moreover, other inflammatory biomarkers [i.e., growth differentiation factor-15 (GDF15), myeloid-related protein 8/14, pentraxin 3, lectinlike oxidized low-density lipoprotein receptor-1 (LOX-1)] have been considered surrogate markers of cardiovascular disease and atherosclerosis in type 2 diabetes patients [13–16].

GDF15 is a member of the transforming growth factor beta family, secreted from cells such as adipocytes and myocytes in response to cellular ischemia and oxidative stress both present in diabetes. GDF15 is a marker of oxidative stress and inflammation and provides independent prognostic information on cardiovascular events [17].

**Figure 1.** Major functions of endothelial cells: regulation of vascular tone, control of VSMC proliferation, inflammation, permeability, angiogenesis, metabolism and hemostasis. Ang II, angiotensin II; CAMs, cell adhesion molecules; CCL; chemokine (C-C motif) ligand; EC, endothelial cell; EDHF, endothelium derived hyperbolizing factor; EGF, epidermal growth factor; ET1, endothelin-1; FGF, fibroblast growth factor; H2 S; hydrogen sulfide; HSPG, heparan sulfate proteoglycans; ICAM, intercellular adhesion molecule; NO, nitric oxide; PAF, platelet-activating factor; PAI-1, Plasminogen activator inhibitor-1; PDGF, platelet-derived growth factor; PGH2 , prostaglandin H2 ; PGI2 , prostacyclin; ROS, reactive oxygen species; TF, tissue factor; TFPI, tissue factor pathway inhibitor; TGF-β, transforming growth factor-β; t-PA, tissue plasminogen activator; TXA2 ; thromboxane A2 ; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cells; vWF, von Willebrand factor.

Myeloid-related protein 8/14 is a heterodimer consisting of two proteins that bind calcium and calgranulin A and B, which play an important role in the signaling pathways of calcium and in the interaction between the cytoskeleton and the membrane [18]. Myeloid-related protein 8/14 is synthesized by activated monocytes and neutrophils and is a pro-inflammatory protein expressed in atherosclerotic plaques associated with atherosclerosis in diabetic patients [19].

Pentraxin 3 is an acute-phase reactant produced by the peripheral tissues at sites of local inflammation and reflects impaired vascular endothelial function [20].

LOX-1 is a *lectin*-*like receptor* for *oxidized low*-*density* lipoproteins (ox-LDL), mainly expressed in endothelial cells, macrophages, smooth muscle cells, and monocytes. This receptor is upregulated by ox-LDL itself and by angiotensin II, endothelin, cytokines, and shear stress. The LOX-1 expressed on the cell surface can be proteolytically cleaved and released in a soluble form (sLOX-1) in the circulation under pathological conditions such as hyperlipidemia and type 2 diabetes [21, 22].

Additionally, galectin-3 might also be an independent marker of vascular remodeling and endothelial dysfunction accompanied by inflammation, proliferation, and atherosclerosis in both normal and diabetic individuals [23, 24]. Galectin-3 is a multifunctional protein that belongs to a family of β-galactoside binding proteins and widely distributes in the heart, brain, visceral **adipose tissue,** and blood vessels. Galectin-3 is able to bind the advanced glycation end products (AGEs) and advanced lipoxidation end products that accumulate in target organs and exert their toxic effects by triggering pro-inflammatory and pro-oxidant pathways [25]. Galectin-3 levels are increased in subjects with obesity and type 2 diabetes [26], and animal studies have suggested that galectin-3 may be involved in the onset and progression of these metabolic disorders by acting primarily at the adipose tissue level. A recent study by Olefsky and co-workers has shown that galectin-3 provides a crucial mechanistic link between inflammation and insulin resistance and that pharmacological inhibition of galectin-3 can increase insulin sensitivity [27].

Inflammation plays a crucial role in the etiology of vascular disease in diabetic states (**Figure 2**). The causes that trigger inflammation are pleiotropic and include most of the features that characterize type 2 diabetes. Arterial hypertension is also a low-grade inflammatory disease [28, 29] often present in diabetes along with hyperinsulinemia, insulin resistance, dyslipidemia, and obesity (**Figure 2**). Chronic exposure to glucotoxicity and lipotoxicity in diabetes induces a pro-inflammatory phenotype in macrophages residing or invading the adipose tissue and the vasculature [30, 31]. The dysfunctional endothelium may enhance leukocyte adhesion and the recruitment of inflammatory cells to the arterial wall, primarily through CCL2, a chemokine that promotes the attraction of immune cells to the sites of inflammation, thereby promoting lipid deposition and facilitating the atherosclerotic plaque formation [28, 32]. In addition, it is well known that the pro-inflammatory transcription factors NFκB and activator protein-1 and kinases such as c-Jun N-terminal kinase, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) are regulated by the cellular redox state [33, 34]. Proatherogenic factors in obesity and diabetes such as oxidized

lipids, angiotensin II, and hyperglycemia increase the activity of NF-κB and MAPKs in endothelial cells and promote the activation of pro-inflammatory cytokines (e.g., IL-6), chemokines (e.g., CCL2, IL-8) [35], the expression of adhesion molecules [intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)] [36] and activation of inducible nitric oxide synthase (iNOS) [37], growth factors, and enzymes [38–40]. The subsequent increment in intracellular ROS production and the activation of the pro-inflammatory signaling complexes—the inflammasomes (including nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome) is responsible for the activation of interleukins such as IL-1β and IL-18, triggering inflammation [41]. The NLRP3 inflammasomes of the innate immune system induce a microinflammatory state stimulating various pro-inflammatory cytokines involved in the pathogenesis of diabetes and

CCL2, chemokine (C-C motif) ligand 2; CCL5, chemokine (C-C motif) ligand 5; COX, cyclooxygenases; CTGF, connective tissue growth factor; CX3CL1, fractalkine; IL- Interleukin; iNOS, inducible nitric oxide synthase; ICAM-1, intercellular adhesion molecule-1; NFκB, nuclear factor κB; TGFβ, *transforming growth factor* β; TLR, toll like receptor; TNF-α; tumor

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its complications.

**Pro-inflammatory cytokines**

**Local inflammation**

Cyclooxygenases—COX Transcription factors as NFκB

**Adhesion molecules**

iNOS

E-selectin **Chemokines** CCL2 (MCP-1) CX3CL1 (fractalkine) CCL5 (RANTES) **Toll-like receptors** Toll like receptor—TLR2 Toll like receptor—TLR4 **Pro-fibrotic factors**

TNF-α; Interleukins IL-1, IL-6, IL-8, IL-22

Intercellular adhesion molecule-1—ICAM-1 Vascular cellular adhesion molecule-1—VCAM-1

Transforming growth factor—TGFβ Connective tissue growth factor—CTGF

necrosis factor α; VCAM-1, vascular cellular adhesion molecule-1.

**Table 1.** Inflammatory components of diabetic complications.


CCL2, chemokine (C-C motif) ligand 2; CCL5, chemokine (C-C motif) ligand 5; COX, cyclooxygenases; CTGF, connective tissue growth factor; CX3CL1, fractalkine; IL- Interleukin; iNOS, inducible nitric oxide synthase; ICAM-1, intercellular adhesion molecule-1; NFκB, nuclear factor κB; TGFβ, *transforming growth factor* β; TLR, toll like receptor; TNF-α; tumor necrosis factor α; VCAM-1, vascular cellular adhesion molecule-1.

**Table 1.** Inflammatory components of diabetic complications.

Myeloid-related protein 8/14 is a heterodimer consisting of two proteins that bind calcium and calgranulin A and B, which play an important role in the signaling pathways of calcium and in the interaction between the cytoskeleton and the membrane [18]. Myeloid-related protein 8/14 is synthesized by activated monocytes and neutrophils and is a pro-inflammatory protein expressed in atherosclerotic plaques associated with atherosclerosis in diabetic

Pentraxin 3 is an acute-phase reactant produced by the peripheral tissues at sites of local

LOX-1 is a *lectin*-*like receptor* for *oxidized low*-*density* lipoproteins (ox-LDL), mainly expressed in endothelial cells, macrophages, smooth muscle cells, and monocytes. This receptor is upregulated by ox-LDL itself and by angiotensin II, endothelin, cytokines, and shear stress. The LOX-1 expressed on the cell surface can be proteolytically cleaved and released in a soluble form (sLOX-1) in the circulation under pathological conditions such as hyperlipidemia

Additionally, galectin-3 might also be an independent marker of vascular remodeling and endothelial dysfunction accompanied by inflammation, proliferation, and atherosclerosis in both normal and diabetic individuals [23, 24]. Galectin-3 is a multifunctional protein that belongs to a family of β-galactoside binding proteins and widely distributes in the heart, brain, visceral **adipose tissue,** and blood vessels. Galectin-3 is able to bind the advanced glycation end products (AGEs) and advanced lipoxidation end products that accumulate in target organs and exert their toxic effects by triggering pro-inflammatory and pro-oxidant pathways [25]. Galectin-3 levels are increased in subjects with obesity and type 2 diabetes [26], and animal studies have suggested that galectin-3 may be involved in the onset and progression of these metabolic disorders by acting primarily at the adipose tissue level. A recent study by Olefsky and co-workers has shown that galectin-3 provides a crucial mechanistic link between inflammation and insulin resistance and that pharmacological inhibition

Inflammation plays a crucial role in the etiology of vascular disease in diabetic states (**Figure 2**). The causes that trigger inflammation are pleiotropic and include most of the features that characterize type 2 diabetes. Arterial hypertension is also a low-grade inflammatory disease [28, 29] often present in diabetes along with hyperinsulinemia, insulin resistance, dyslipidemia, and obesity (**Figure 2**). Chronic exposure to glucotoxicity and lipotoxicity in diabetes induces a pro-inflammatory phenotype in macrophages residing or invading the adipose tissue and the vasculature [30, 31]. The dysfunctional endothelium may enhance leukocyte adhesion and the recruitment of inflammatory cells to the arterial wall, primarily through CCL2, a chemokine that promotes the attraction of immune cells to the sites of inflammation, thereby promoting lipid deposition and facilitating the atherosclerotic plaque formation [28, 32]. In addition, it is well known that the pro-inflammatory transcription factors NFκB and activator protein-1 and kinases such as c-Jun N-terminal kinase, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) are regulated by the cellular redox state [33, 34]. Proatherogenic factors in obesity and diabetes such as oxidized

inflammation and reflects impaired vascular endothelial function [20].

patients [19].

and type 2 diabetes [21, 22].

234 Endothelial Dysfunction - Old Concepts and New Challenges

of galectin-3 can increase insulin sensitivity [27].

lipids, angiotensin II, and hyperglycemia increase the activity of NF-κB and MAPKs in endothelial cells and promote the activation of pro-inflammatory cytokines (e.g., IL-6), chemokines (e.g., CCL2, IL-8) [35], the expression of adhesion molecules [intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)] [36] and activation of inducible nitric oxide synthase (iNOS) [37], growth factors, and enzymes [38–40]. The subsequent increment in intracellular ROS production and the activation of the pro-inflammatory signaling complexes—the inflammasomes (including nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome) is responsible for the activation of interleukins such as IL-1β and IL-18, triggering inflammation [41]. The NLRP3 inflammasomes of the innate immune system induce a microinflammatory state stimulating various pro-inflammatory cytokines involved in the pathogenesis of diabetes and its complications.

In endothelial cells, vascular smooth muscle cells, monocytes and macrophages, the intracellular synthesis of diacylglycerol is increased in hyperglycemia, leading to the activation of the PKC pathway [45, 46]. In monocytes, there is a subsequent release of the integrins CD11b, CD11c, and CD14 [47, 48]. CD11b or CD11c receptor occupation on the surface of human monocytes stimulates cell-specific pro-inflammatory pathways such as secretion of IL-8, macrophage inflammatory protein (MIP)1α and MIP1β [49]. CD14 + CD16+ monocytes are also

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Hyperglycemia also upregulates toll-like receptor (TLR) activity through an increment in ROS augmenting inflammation. In human monocytes, Dasu and colleagues [51] reported that high glucose induces TLR2 and TLR4 expression through PKC activation, by stimulating NADPH oxidase (NOX). Several other studies have demonstrated that under hyperglycemic conditions, reducing ROS and specifically NOX activity reduced TLR expression and activity [52, 53].

AGEs are generated in vivo as a normal consequence of metabolism, but their formation is accelerated under conditions of hyperglycemia, hyperlipidemia, and increased oxidative stress [54–57]. AGEs are highly reactive and can trigger inflammation by generating particularly TNF-α and IL-6 [58]. In addition, AGEs activate their receptors/binding sites (RAGE and lactoferrin-like polypeptide complex) in endothelial cells, monocytes, and macrophages leading to the activation of MAPK and NF-κB. AGEs also enhance the formation of oxidized low-density lipoprotein (oxLDL) and during hyperglycemia the expression of LOX-1 on monocytes and macrophages increases. These processes further facilitate the uptake of oxLDL

Another important mechanism to cause hyperglycemia-induced endothelial dysfunction is the redox-dependent activation of endothelial NLRP3 inflammasomes [61]. Endothelial tight junction disruption in diabetes requires NLRP3 inflammasomes. High glucose activates NLRP3 inflammasome in endothelial cells via ROS production. Reducing ROS production abolished high glucose-induced inflammasome activation, tight junction disruption, and endothelial hyperpermeability in endothelial cells. The clinical potential of targeting inflammasome sig-

Lipids also induce a state of inflammation. In diabetes, lipids increment the inflammatory process by promoting oxidative stress and leukocyte activation and ultimately foster endothelial dysfunction and atherosclerosis progression. The ingestion of high fat diets results in increased leukocyte activation, which is reflected by an increase of surface expression of CD11b, CD11c and CD14 on monocytes and CD11b, CD66b and CD62L on neutrophils [47, 62, 63]. These results suggest a pro-inflammatory effect of dietary lipids on circulating inflammatory cells with detrimental effects on the vessel wall. After a meal, the remnants of triglyceride-rich lipoproteins and *oxLDL* are taken up by circulating leukocytes, macrophages, endothelial cells, and smooth muscle cells, activating the PKC pathway and resulting in *NF-κB* activation [64–66]. NF-κB promotes the transcription of various inflammatory genes, including genes encoding for cytokines, chemokines, and adhesion molecules [59]. In addition, FFA and cholesterol induce inflammation by activating TLR pathways and, subsequently,

naling axis for prevention of the early onset of diabetic vasculopathy is evident [61].

linked with pro-inflammatory conditions [50].

by macrophages, thus increasing inflammation [59, 60].

*2.2.2. Lipids-induced inflammation*

**Figure 2.** Risk factors for endothelial dysfunction associated with type 2 diabetes. Major role for oxidative stress and inflammation. AGEs, advanced glycation end products; FFAs, free fatty acids.
