Roles of LOX-1 in microvascular dysfunction

    Roles of LOX-1 in microvascular dysfunction Valter Lubrano, Silvana Balzan PII: DOI: Reference:

S0026-2862(16)30012-7 doi: 10.1016/j.mvr.2016.02.006 YMVRE 3608

To appear in:

Microvascular Research

Received date: Revised date: Accepted date:

23 November 2015 17 February 2016 17 February 2016

Please cite this article as: Lubrano, Valter, Balzan, Silvana, Roles of LOX-1 in microvascular dysfunction, Microvascular Research (2016), doi: 10.1016/j.mvr.2016.02.006

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ACCEPTED MANUSCRIPT Roles of LOX-1 in microvascular dysfunction Valter Lubrano1 and Silvana Balzan2 Fondazione CNR/Regione Toscana G. Monasterio, Pisa, Italy

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Institute of Clinical Physiology, CNR, Pisa, Italy

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Corresponding author: Dr. Valter Lubrano

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Fondazione CNR/Regione Toscana G. Monasterio Via Moruzzi n° 1

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56100 - Pisa, Italy

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e-mail: [email protected]

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Tel. +390503152199; Fax +39-050-3153454

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Abstract

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Studies from human and animal models with metabolic disease and hypertension highlight atrophic

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remodeling, reduced lumen size and thinner vascular walls of microvessels with profound density

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reduction. This impaired vascular response limits the perfusion of peripheral tissues inducing organ damage. These conditions are strongly associated with oxidative stress and in particular with the up-

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regulation of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Several factors such as cytokines, shear stress, and advanced glycation end-products, especially oxLDL, can up-regulate

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LOX-1. The activation of this receptor induces the production of adhesion molecules, cytokines and the release of reactive oxygen species via NADPH oxidase. LOX-1 is considered a potent mediator

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of endothelial dysfunction and it is significantly associated with reduced microvascular

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endothelium NO-dependent vasodilation in hypercholesterolemia and hypertension. Microvascular

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endothelial cells increased the expression of IL-6 in association with the increased concentration of LDL and its degree of oxidation. Moreover, increased IL-6 levels areassociated with up-regulation

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of LOX-1 in a dose-dependent manner. Another consequence of microvascular inflammation is the generation of small amounts of ROS, similar to those induced by low concentration of oxLDL (< 5 g /mL) which induces capillary tube formation of endothelial cells, through LOX-1 up-regulation. In light of its central role, LOX-1 represents an attractive therapeutic target for the treatment of human atherosclerotic diseases and microvascular disorders.

Key words: oxLDL receptor, LOX-1, free radicals, oxidative stress, endothelial function, microcirculation

ACCEPTED MANUSCRIPT Introduction Microcirculation appears to have a relevant role in the genesis of target organ damage in various

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diseases such as diabetes, hypertension and dyslipidemia. Animal models affected by metabolic

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syndrome are characterized by atrophic remodeling, reduced lumen size and thinner vascular walls

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of the microvasculature, which occurs before the onset of diabetes and in the absence of atherosclerotic lesions (Stepp et al., 2004). There is also a profound reduction in microvessel

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density, which negatively influences transport and exchange with parenchymal tissues (Frisbee, 2005) (fig 1). In addition, in diabetic patients with metabolic syndrome, an increment of circulating

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endothelin-1 activity and of the vasoconstrictor tone have been reported (Cardillo et al., 2002). This impaired vascular response could limit the perfusion of peripheral tissues; infact metabolic

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syndrome is characterized by inadequate perfusion in muscle with elevated metabolic requirements

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(fig. 1)

Fig. 1 Links between metabolic diseases and vascular abnormalities leading to mortality

ACCEPTED MANUSCRIPT Effect of metabolic disease and hypertension on microcirculation Coronary flow reserve (CFR) measured by trans-thoracic Doppler echocardiography (TTDE) has

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been introduced as a reliable and reproducible indicator of coronary microvascular-endothelial

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function. These analysis suggest that coronary microvascular-endothelial dysfunction is present in

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patients affected by isolated systolic hypertension (Bozbas et al., 2012) and in type 2 diabetic patients showing renal, ocular or both, microvascular complications (Avogaro et al., 2007). The

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presence of these microvascular dysfunctions has been shown to be strongly associated with oxidative stress. One of the mechanisms of oxidative stress that might promote and chronically

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sustain microcirculatory dysfunction is the interaction between LDL lipoproteins and the endothelium. In this respect, modification of LDL induced by oxidative stress appears to play the

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major role. Oxidative stress depresses endothelium-dependent vasodilation and causes endothelial

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cell apoptosis through impairment of eNOS synthesis (Edirisinghe et al., 2008). Oxidized LDL

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(oxLDL) can interact with vascular tissues either by a nonspecific propagation of oxidative insult or through specific receptors. Both oxLDL and advanced glycosylation end products (AGEs) upregulated the expression of the oxLDL receptor-1 (LOX-1) (Fig. 2), which leads to enhanced

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accumulation and adhesion of inflammatory cells, including dendritic cells, macrophages and leukocytes, in vascular endothelium (Ge et al., 2005; Shiu et al., 2012). These events induced by oxidative stress and inflammatory cells are also observed in patients affected by systemic sclerosis and might contribute to maintain microvascular endothelium injury (Maugeri N et al., 2014)

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LOX-1

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Fig 2 LOX-1 modulation by metabolic dysfunction and hypertension

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LOX-1 was first cloned as a major receptor for oxidatively modified LDL by Sawamura and colleagues (Sawamura et al., 1997) and it has recently been reported that it accepts oxLDL as a ligand, binding and internalizing it by receptor-mediated endocytosis (Twigg et al., 2012). It has a type II membrane protein structure with a short N-terminal cytosolic domain and a long C-terminal extracellular domain, which is distinct from the type 1 and 2 scavenger receptors, CD36 and CD68 (Sawamura et al., 1997). It has been suggested that oxLDL uptake through LOX-1 receptor, abundantly present on the surface of vascular endothelium, might be involved in endothelial activation or dysfunction (Goyal et al., 2012). Several factors such as cytokines, shear stress, and advanced glycation end-products can also upregulate LOX-1 (Jono et al., 2002; Li et al., 1999; Murase et al., 1998). LOX-1 expression is up-

ACCEPTED MANUSCRIPT regulated in pathological conditions, such as hypertension, diabetes and hypercholesterolemia (Chen et al., 2001; Nagase et al., 1997; Chen et al., 2000). Activation of LOX-1 by oxLDL induces up-

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regulation of monocyte chemotactic factor (MCP)-1, intercellular adhesion molecule (ICAM)-1,

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vascular cell adhesion molecule (VCAM)-1 and the release of reactive oxygen species via NADPH

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oxidase (Li et al., 2000: Cominacini et al., 2001; Cominacini et al., 2000; Mehta et al., 3001). Moreover, the binding of several ligands to LOX-1 induces superoxide generation, inhibit nitric oxide production (NO), enhances endothelial adhesiveness of leukocytes and induce chemokines

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expression (Li et al., 2000: Cominacini et al., 2001; Cominacini et al., 2000). Among the

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polymorphic forms of LOX-1 gene (OLR1) that contribute to modify the susceptibility to cardiovascular diseases, the variant form of LOX-1, LOXIN, lacking of the functional domain,

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appears to play an important role.

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This form showed a protective function for LOX-1 activation due to direct interaction with it

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(Mango R. et al., 2011). In fact, recent findings have shown that the overexpression of LOXIN protects endothelial progenitor cells from apoptosis induced by oxLDL (Veas C. et al., 2016).

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LOX-1 and NO dependent vasodilation in microvascular endothelium Microvascular dysfunction involving a reduction of NO dependent vasodilation is clearly evident in cutaneous circulation, especially in hypercholesterolemia (Holowatz LA et al., 2011; Rossi et al., 2009). Cutaneous NO-dependent vasodilation, in response to an endothelial NO synthase (eNOS)specific stimulus, is attenuated in human subjects with LDL>160 mg/dL (Kenney et al 2013). Several hypothesis have been put forward by various authors regarding the reduction of NO bioavailability induced by hypercholesterolemia, including the upregulation of arginase activity which limits eNOS substrate availability (Holowatz LA et al., 2011) or a reduction in the essential eNOS cofactor tetrahydrobiopterin (BH4) (Holowatz LA and Kenney WL, 2011).

ACCEPTED MANUSCRIPT Serum sLOX-1 concentration, an indirect measure of LOX-1 receptor, was found to be significantly associated with reduced NO-dependent vasodilation in hypercholesterolemic patients (Kenney et al

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2013) (Fig. 3). LOX-1 has been shown to be activated by shear stress, endothelin-1 (ET-1),

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angiotensin II (Angio II) and oxidative stress. The binding of oxLDL to LOX-1 activates the

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membrane multi-subunit enzyme NADPH oxidase on endothelial cells, resulting in a rapid increase of intracellular reactive oxygen species (ROS) (Fig. 3). Generated ROS include superoxide anion (O2-), hydrogen peroxide (H2O2), peroxynitrite (ONOO-), NO, and hydroxyl (OH-) radicals. In

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particular, O2- is responsible for inactivating NO in a chemical reaction during which the cytotoxic

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radical peroxynitrite is formed (Ignarro, 1990; Huie and Padmaja, 1993) (Fig. 3). Increased O2- not only reacts with intracellular NO, but also up-regulates LOX-1 expression, thereby contributing to

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further ROS generation (Chen ET AL., 2007; Cominacini et al., 2000) (Fig 3).

Fig. 3 Biochemical pathways involved in hypercholesterolemia and endothelial dysfunction.

ACCEPTED MANUSCRIPT Our recent data showing the effects of free radicals on LOX-1 upregulation in Human Microvascular Endothelial Cells (HMEC-1) suggest that ROS may act alone, without the presence

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of native LDL in LOX-1 upregulation (Lubrano and Balzan, 2014). Moreover, in the same cells we

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observed a dose related increment of LOX-1 levels after treatments with increased doses of IL-6, an

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important finding showing the adverse effect induced by inflammation in microcirculation (Lubrano et al., 2015).

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We demonstrated that hypercholesterolaemia is associated with elevated plasma concentrations of both NOx (NO2/NO3, NO metabolites) and MDA and that the effective cholesterol lowering

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therapy with atorvastatin normalizes these two parameters (Fig. 3). In vitro, both in HMEC-1 and umbilical vein endothelial cells, mildly oxLDL enhances the synthesis of eNOS and promotes the

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generation of NOx (Lubrano et al., 2003). These data were supported by the demonstration that the

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aortic ring of high-cholesterol-fed rabbits with a reduced response to acetylcholine showed a two-

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fold greater production of NO both at baseline and in response to endothelium-dependent vasodilators (Minor et al., 1990). An impaired endothelium-dependent (i.e. NO mediated) vascular relaxation, coupled with a normal response to NO precursors, both in resistance arterioles and in

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conduit arteries, has consistently been found in dyslipidemic patients and has been interpreted as evidence of endothelial dysfunction. These apparently inconsistent results can be explained by the demonstration that oxidized lipid substrates, by interacting with a specific cell receptor LOX-1, rapidly increased intracellular reactive oxygen species and decreased intracellular NO concentration (Cominacini et al., 2001; Lubrano and Balzan, 2014); in this context O2- is known to inactivate NO in a chemical reaction during which peroxynitrite is formed (Lubrano and Balzan, 2014). The degree of oxidative stress could induce two functionally different forms of eNOS, the coupled and uncoupled ones, depending on the availability of the cofactor tetrahydrobiopterin. The uncoupled form is not able to generate NO and directly produces ROS and peroxynitrites. The increment of oxidative stress could reduce NO level by presence of uncoupled eNOS or inactivation of NO.

ACCEPTED MANUSCRIPT Therefore, the less efficient generation of biologically active NO and the increment of lipoperoxides could stimulate an up-regulation of eNOS production and activity to counterbalance the reduced NO

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bioavailability.

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Products from metabolic disorders can amplify the oxidative stress by inducing LOX-1and an

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excessive bioavailability of ROS, with the onset of an insufficient antioxidant defense, resulting in an imbalance between the production and destruction of ROS. The normal production of NO is

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important in microcirculation and the presence of ROS can influence normal capillary tissue development and function. Indeed it was shown that inhibition of NOS abolished exercise-induced

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capillary angiogenesis in skeletal muscle (Hudlická et al., 2000). Regulation of VEGF gene expression induced by NO has been demonstrated in numerous cell types, including vascular

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smooth muscle cells (Dulak et al., 2000).

A demonstration of the fact that lipoperoxides favor the production of LOX-1 and depletion of NO

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is the observation that lipoprotein apheresis reduced the expression of pro-atherosclerotic oxLDL receptor LOX-1 and adhesion molecule VCAM-1 and increased the production of NO in human

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endothelial cells in response to serum from hypercholesterolemic patients (Morawietz et al., 2013). The reduced presence of NO was also observed in subjects affected by hypertension (Fig 4). Ang II causes vasoconstriction by activation of the predominant AT1 receptor in vascular smooth muscle cells. Furthermore, Ang II increases vascular NADPH oxidase expression and superoxide anion formation (Cai et al., 2003). O2- can reduce vascular NO availability by peroxynitrite formation leading to endothelial dysfunction (Cai H, Harrison DG, 2000). Ang II upregulates LOX-1 gene expression and oxLDL up-regulates Ang II type 1 receptors (AT1R) expression in cultured HCAECs (Li et al., 2000) (Fig. 4). Angiotensin-converting enzyme (ACE) inhibitors and AT1R blockers decrease LOX-1 expression (Morawietz et al., 1999). Previous studies have shown that AT1R activation stimulates the expression of LOX-1 (Li et al., 2000) and LOX-1 activation, in turn,

ACCEPTED MANUSCRIPT upregulates AT1R expression. Activation of both AT1R and LOX-1 induces a state of oxidative

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stress (Mehta et al., 2006).

Fig. 4 Biochemical pathways involved in hypertension and endothelial dysfunction

LOX-1 and microvascular inflammation Serum markers of inflammation have been identified as risk markers of cardiovascular disease. Highly sensitive C-reactive protein (CRP) has proven to be a strong predictor of cardiovascular events (Shah , Newby , 2003; Ridker et al., 2000). It has been shown that CRP increases the release

ACCEPTED MANUSCRIPT of inflammatory cytokines (Ballou, Lozanski, 1992), enhances the binding of monocytes to the endothelium (Woollard et al., 2002) and favors macrophage foam cell formation (Fu, Borensztajn,

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2002). CRP also decreases eNOS activation while it increases the expression of endothelial cell

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adhesion molecules, chemokines, ET-1 and plasminogen activator inhibitor-1 (Pasceri et al., 2001;

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Verma et al., 2002). The influence of both native and oxidized LDL on the release of potent vasoactive substances, such as NO and ET-1, by endothelium is potentially relevant both in

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initiating and maintaining abnormal tissue perfusion (Lu et al., 2005).

In vitro studies showed that endothelial LOX-1 expression is induced by various pro-inflammatory

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cytokines, such as tumor necrosis factor-alfa (TNF-alfa) (Kume et al., 1998) and transforming growth factor-1 (TGF-1) (Draude et al., 2000), as demonstrated for pro-atherogenic factors, such as

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oxLDL and AGEs (Chen et al., 2001). Vascular inflammation and receptor up-regulation, events

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associated with atherosclerosis, represent important factors that improve the uptake of LDL and ox-

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LDL.

Our recent finding indicates that in microvascular endothelial cells, the expression of IL-6 increased

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in a dose dependent manner in the presence of moderate to high levels of LDL and oxidation, . The incubation of the cells with IL-6 induced an evident up-regulation of LOX-1, as previously mentioned (Lubrano et al., 2015). Other studies indicated that also soluble IL-6 receptor (sIL-6R gp80 ), by binding IL-6, could induce LOX-1 in HAEC (Li et al., 2004). LOX-1 is particularly responsible for the reduction of oxLDL from the blood. Its expression is induced by two factors, the increase of oxLDL and the presence of inflammation. This biological mechanism could explain the rapid reduction of cholesterol during the acute phase of inflammatory disease, considering that the cells of microcirculation are representative of the entire circulatory system. Our data indicate that endothelial cells, trying to reduce the high plasma levels of oxLDL, cause injury to endothelium (Lubrano et al., 2015).

ACCEPTED MANUSCRIPT In coronary artery disease, the diffuse inflammation seems to be the major determinant of clinical instability, more than the vulnerable plaque inflammation. Indeed, after acute ischemic injury,

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inflammatory alterations of microvessels may be early predictors of an adverse cardiac outcome

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(Eagle et al., 2010). Interactions between oxLDL and its receptor, LOX-1, appear to play a key role

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in oxLDL-induced vascular dysfunction, including cell apoptosis and matrix metalloproteinase (MMP) production and activation, which evoke atherosclerotic plaque rupture or erosion (Kunjathoor et al., 2002; Kita et al., 2001). Moreover, oxLDL/LOX-1 receptor interaction

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modulates cellular function (Cominacini et al., 2000) and induces NFkB activation in human

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endothelial cells and in monocytes of unstable angina (UA) patients. This activation promotes the expression of genes involved in the immune-mediated inflammatory reaction in UA. The

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transcription factor octamer (Oct)-1 plays a major role in oxLDL induced LOX-1 promoter

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activation in human endothelial cells, especially when oxLDL and Ang II concur in inducing LOX-1 expression such as in UA microvessel inflammation (Neri et al., 2003; Neri et al., 2004; Chen et al.,

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2006). Coronary atherosclerosis must not be considered a disease limited to the large and mediumsize arteries but also extended to the microvasculature. Microvessel inflammation shows several

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features peculiar of atherosclerosis such as endothelial cell activation, apoptosis or proliferation, the presence of dendritic positive cells as well as of endothelial cells and macrophages containing oxLDL. Apoptosis is a deleterious event which can lead to a structural rarefaction and thinning of the endothelium causing elevation of microvascular resistance (Greene, 1998). In patients affected by UA, a marked accumulation of oxLDL was found in inflamed microvessels, contributing to the immune-mediated component of UA inflammatory process (Neri, 2013). The other consequence of microvascular inflammation is the generation of small amounts of ROS, like those induced by low concentrations of oxLDL(< 5 g/mL), that appears to stimulate angiogenesis through up-regulation of LOX-1, and the LOX-1-mediated activation of NADPH oxidase/mitogenactivated protein kinases/NF-κB pathway. Small concentrations of oxLDL induce vascular

ACCEPTED MANUSCRIPT endothelial growth factor (VEGF), which is the main factor responsible of capillary tube formation in endothelial cells (Dandapat et al., 2007). This event may represent a pathological sign of

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prelaminar endothelial activation which over time can lead to major vascular diseases.

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The increase in cortical vascular density found in hypercholesterolemic pigs was attenuated after

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antioxidant vitamin treatment with corresponding decreases in the cortical tissue levels of VEGF and immunostaining for the VEGF receptor. Likewise, interstitial fibrosis and inflammation

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accompanying hypercholesterolemic diet were attenuated by antioxidant vitamin supplementation

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(Chade et al., 2004).

Some authors indicated a possible inhibitory cytokine therapy in patients affected by vascular disorders. Zhang H et al., (2009) suggested a chronic anti-inflammatory treatment with anti-TNF-α

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antibody that showed to improve endothelial function of brachial artery in patients with rheumatoid arthritis, systemic vasculitis and Crohn’s disease. Unfortunately the vascular inflammatory disease

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is caused by various cytokines, and it is therefore difficult to perform an inhibitory therapy against the numerous cytokines causing vascular diseases. In the presence of risk factors such as

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hypertension, diabetes, dyslipidemic states, metabolic syndrome, LOX-1 based therapy may be used to prevent the oxLDL binding and the oxidation of LDL-cholesterol. LOX-1 inhibition, using antibody or knockout strategy would inhibit endothelial dysfunction and consequently the vascular inflammation. Some authors showed that this therapy clearly reduced the extent of myocardial injury and the subsequent cardiac remodeling (Li et al., 2003; Hu et al., 2008). Experimental evidence of LOX-1 effects on some microvascular beds. OxLDL and LOX-1 may play a role in the increased inflammation and capillary leakage, two factors which induce disturbances in the microcirculation, contributing to the development of sepsis and multiorgan failure. For this reason, some authors examined the role of oxLDL and LOX-1 in different microcirculatory pathological districts. In intestinal inflammation the microhemodynamic

ACCEPTED MANUSCRIPT parameters were related to oxLDL or LOX-1 expression (Lehr et al., 1993). Administration of antibodies against LOX-1 significantly reduced endotoxin-induced leukocyte adherence to intestinal

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submucosal venules. Moreover LOX-1 was reduced significantly both at mRNA and protein levels.

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MCP-1 plasma levels were found to be decreased after administration of antibodies against LOX-1

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(Landsberger et al., 2010). In hamsters or in endotoxemic rats treated with LOX-1 antibodies and exposed to oxLDL, the capillary microperfusion was not affected. Instead, in isolated microvessels

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exposed to oxLDL, a reduction of oxidative stress induced endothelial-dependent vasodilation was observed and it could be reversed by incubation with oxygen radical scavengers (Lehr et al., 1993;

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Landsberger et al., 2010).

Alterations in cutaneous vascular signaling are evident in early processes of atherosclerosis and are

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coronary (Khan et al., 2008) and renal circulations (Coulon et al., 2012; Debbabi et al., 2010) . Hypercholesterolemic patients exhibited a significantly attenuated cutaneous vasodilatory response

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to local skin heating, a stimulus known to induce vasodilation predominantly through the production of NO via eNOS (Bruning et al., 2012; Kellogg et al., 2008). These findings are supported by our un-published data observed in 12 hypercholesterolemic patients (TC=320±77; HDL=53.14±13; TG=118.1±56.6 and LDL=242.6±85.3 mg/dl; age 50±12 years) on cutaneous NO-dependent vasodilation in response to atorvastatin therapy. Measuring the NO products levels and hand microcircolatory flow (MF) by laser Doppler technique, we did not observed any correlation between MF and NOx production at baseline condition, whereas it was observed after pharmacological reduction of LDL by atorvastatin (r2=0.33; p<0.05) (Fig 5a, 5b).

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Fig. 5 Regression analysis of the association between NOx plasma levels and hand MBF of (a) hypercholesterolemic patients before therapy and (b) after 6 weeks of therapy with atorvastatin. UA, Arbitrary Unit

As previously documented, the presence of high levels of oxLDL in hypercholesterolemia are responsible of the up-regulation of LOX-1 (Lubrano and Balzan, 2014) that increases the production of free radicals, especially O2-, contributing to NO depletion . In our study, the therapy restored the normal levels of NO, so that a significant relationship with MF, as in normal subjects, was observed. Some authors (Rossi et al., 2009) showed that changes in skin microvasculature may precede and predict vascular dysfunction in larger systemic vessels and that there was a significant

ACCEPTED MANUSCRIPT relation between NO-dependent vasodilation in cutaneous microvasculature and serum LDL, oxLDL cholesterol, sLOX-1 receptors, and triglycerides. LOX-1 stimulation activates multiple

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downstream signaling events leading to decrease of NO bioavailability through NADPH oxidases

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(Mitra et al., 2011) and the increase of arginase activity thus contributing to NOS uncoupling (Ryoo

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et al., 2011). The interaction between leukocytes and endothelium in presence of oxLDL or LOX-1 has been studied both in vitro and in vivo. Surface coating with LOX-1 was sufficient to induce the adhesion of polymorphonuclear cells (PMNs) under physiologic shear conditions (Honjo et al.,

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2003). The block of LOX-1 had an inhibitory effect on leukocyte adhesion. In animals with

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endotoxemia the retinal microvessels appear to have reduced interaction with leukocytes after LOX1inhibition. This study therefore suggested that LOX-1 is a vascular specific ligand (Honjo et al.,

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2003).The association between increasing LOX-1 expression and microvascular flow disturbance is

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documented by patients without coronary artery disease that sometimes develop chest discomfort associated with the occurrence of atrial fibrillation (AF). Some studies have clearly shown that AF

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increases systemic Ang II levels (Goette et al., 2008; Cardin et al., 2003). Ang II upregulates LOX-1 gene expression and oxLDL up-regulates AT1R expression (Li et al., 2000), generating ROS; in

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turn ROS can rapidly react with nitric oxide NO, leading to peroxynitrite formation, reduced NO availability, and endothelial dysfunction (Doughan et al., 2008; Oudot et al., 2003). The increase of LOX-1 receptor in the present model and its prevention by blocking AT1-receptors resembled the Ang II-mediated up-regulation of LOX-1 in human endothelial cells (Morawietz et al., 1999). Taken together, the AT1-receptors block and the reduction of Nox2, LX-1, and F2-isoprostane effectively prevent the microvascular flow abnormalities and may contribute to the effectiveness of AT1inhibitors to halt the progression of ventricular remodeling (Goette et al., 2009).In some cases, when the intensity of oxidative stress is low, the increase of LOX-1 expression could involve neovascularization, as previously described (Lubrano and Balzan, 2014). Indeed in pig some authors reported that the experimental hypercholesterolemia induced renal cortical neomicrovascularization

ACCEPTED MANUSCRIPT was associated with inflammation. In fact it was observed that the LOX-1 increase was associated with renal inflammation, fibrosis, upregulation of VEGF and its receptor Flk-1, likely mediated by

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increased endogenous oxidative stress. Chronic antioxidant supplementation may preserve the

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kidney of hypercholesterolemic pig (Chade et al., 2004).

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LOX-1 therapy against atherosclerosis and microvascular damage

It may be possible to develop strategy to prevent atherosclerosis and microvascular damage directed

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against major risk factors like metabolic disease and hypertension. Unfortunately pharmacological

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treatment is not efficient and therefore would be important to introduce other strategies. Increasing numbers of studies showed that LOX-1 is a scavenger receptor and is regarded as a central element

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in the initiation of endothelial dysfunction and its further progression to atherosclerosis.

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It is necessary to develop therapeutic strategies that regulate the production of LOX-1 or prevent its

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link with oxLDL. Administration of anti-LOX-1 antibodies inhibits atherosclerosis by decreasing endothelial dysfunction. For example, administration of an anti-LOX-1 neutralizing antibody reduced infarction area, brain edema and apoptotic cell death (Akamatsu et al., 2014). Over the past

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decade, multiple drugs including naturally occurring antioxidants, statins, anti-inflammatory agents, antihypertensive and antihyperglycemic drugs have been shown to inhibit vascular LOX-1 expression and activity. Therefore, LOX-1 represents an attractive therapeutic target for the treatment of human atherosclerotic diseases and microvascular disorder. The central role of this receptor on endothelial damage is highlighted by the fact that the allelic variant "LOXIN", the isoform lacking part of the functional domain, exon 5, when expressed in absence of LOX-1, shows a diminished plasma membrane localization and a deficiency in oxLDL ligand binding. However, the effects of overexpressing LOXIN in the absence of LOX-1 overexpression is non physiological and not well known. Experimental evidence in an in vivo model showed that LOXIN is capable of inhibiting the development of atherosclerosis induced by

ACCEPTED MANUSCRIPT LOX-1 overexpression (White SJ et al., 2009). Other studies showed that the increase of LOXIN expression was linked to a lower rate of acute coronary syndromes (Mango et al., 2015). When

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LOX-1 and LOXIN are co-expressed, they interact forming multimeric protein complexes that lead

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to a significant down-regulation of surface LOX-1 receptors with consequent impairment of oxLDL

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binding and uptake (Biocca et al., 2008). The fact that LOXIN exerts a negative effect on LOX-1 function, makes it an attractive new target for prevention and treatment of atherosclerosis and

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Acknowledgement

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microcirculation damage.

The authors are grateful to Dr. Lucrecia Mota Garcia, Dr. Marcella Simili and Dr. Mike Minks for

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their English editing support. Declaration of interest

The authors declare that they have no conflict of interest

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Highlights  Metabolic syndrome is characterized by reductions in microvessel density and endothelial dysfunction.  LOX-1 up-pregulation by oxLDL induces endothelial dysfunction.  Microcircolatory flow and NO correlate in hypercholesterolemics after therapy.  LOX-1 represents a therapeutic target for microvascular disorders