Accepted Article

DR. DANIELE MANCARDI (Orcid ID : 0000-0003-3809-6047)

Article type : Review

Endothelial dysfunction and cardiovascular risk in lupus nephritis: new roles for old
players?

Daniele Mancardi1, ElisaArrigo1, Martina Cozzi2,3, Irene Cecchi2, Massimo Radin2, Roberta
Fenoglio2, Dario Roccatello2, and Savino Sciascia2.

1 Department of Clinical and Biological Sciences, University of Torino, Italy.

2 Center of Research of Immunopathology and Rare Diseases-Nephrology and Dialysis S.
Giovanni Bosco Hospital, Department of Clinical and Biological Sciences, University of Turin,
Italy.
3 School of Specialization in Nephrology, University of Verona

Corresponding Author:
Savino Sciascia, MD, PhD
Center of Research of Immunopathology and Rare Diseases- Coordinating Center of Piemonte and
Valle d’Aosta Network for Rare Diseases, and SCDU Nephrology and Dialysis, Department of
Clinical and Biological Sciences, S. Giovanni Bosco Hospital and University of Turin, Italy.
Piazza del Donatore di Sangue 3, 10154, Torino, Italy.
Email savino.sciascia@unito.it, Tel +390112402056 Fax +390112402052
Funding: MR is funded by a grant from the Italian Ministry of Health SG-2018-12368028

Running title: News on endothelium and Lupus Nephritis

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/eci.13441
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 Abstract.
Accepted Article
In Systemic Lupus Erythematosus (SLE) patients, most of the clinical manifestation share a
vascular component triggered by endothelial dysfunction. Endothelial cells (ECs) activation
occurs both on the arterial and venous side, and the high vascular density of kidneys accounts for
the detrimental outcomes of SLE through lupus nephritis (LN). Kidney damage, in turn, exerts a
negative feedback on the cardiovascular (CV) system aggravating risk factors for CV diseases
such as hypertension, stroke and coronary syndrome among others. Despite the intensive
investigation on SLE and LN, the role of endothelial dysfunction, as well as the underlying
mechanisms, remains to be fully understood, with no specifically targeted pharmacological
treatment. It is not known, in fact, if the activation pathway(s) in venous ECs are similar to the one
in arterial ECs and doubts persist on the shared manifestation of microcirculation compared to
macro-circulation. In this work, we aim to review the recent literature about the role of endothelial
activation and dysfunction in the development of CV complications in SLE and LN patients. We,
therefore, focus on arterio-venous similarities and differences and on specific pathways of great
vessels compared to capillaries. Critically summarising the available data is of pivotal importance
for both basic researchers and clinicians in order to develop and test new pharmacological
approaches in the treatment of basic components of SLE and LN.

Keywords: Lupus Erythematous, Nephritis, Endothelial Dysfunction;

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 Systemic Lupus Erythematosus, Lupus Nephritis and cardiovascular events: clinical
Accepted Article
perspective
Systemic lupus erythematosus (SLE) is a chronic autoimmune condition characterized by
heterogeneous clinical futures. Over the years, the progression in therapy in SLE has been
paralleled by an improvement in the life expectancy[1,2]. Nevertheless, one of the challenges in
the management of patients with SLE relies on the optimal control of cardiovascular (CV) risk. In
fact, patients with SLE are known to have an increased risk of cardiovascular events (CVE), due
to both traditional and disease-specific risk factors, including inflammation, endothelial
dysfunction, accelerated atherosclerosis and lupus nephritis (LN)[3,4].To date, CVE represent the
most common cause of death in SLE, with CV and cerebrovascular diseases having a tremendous
burden of morbidity [5].Lupus nephritis itself leads to increased risk for CVE. This condition,
indeed, comprises a variety of disease-specific detrimental factors that further increase CV risk.
These include well-recognized factors, such as proteinuria, nephrotic syndrome, prolonged use of
glucocorticoids, higher prevalence of arterial hypertension, dyslipidaemia, chronic kidney disease
(CKD), end stage renal disease (ESRD), but also other less-defined players, endothelial
dysfunction, injury, and inflammation. It has been shown that LN patients are much more prone to
accelerated atherosclerosis than non-LN SLE patients [6]. Lupus nephritis has an additional (up to
8.5-fold) CV risk when compared with non-renal lupus [7,8], consequently leading to an overall
increased mortality [9]. When analysing the CV risk of SLE patients in a population-based case-
control study, Wells and Ward [8] found that the LN was associated with a 2.8-fold increased risk
of acute myocardial infarction. In a subsequent Danish population-based cohort study involving
1644 SLE patients, Hermansen and co-workers showed that the presence of LN confers an 8-fold
increased CV mortality when compared with non-renal SLE patients [7]. Interestingly, when
focusing on the type of renal involvement, as described by specific classes of LN, Sun at al. [10]
showed a trend for an increased CV risk associated with proliferative LN, with the concomitant
presence of class III/V LN doubling the risk for CVE when compared to SLE patients without LN.
Taken the above considerations together, while it is well established that LN leads to increased
CV risk, higher than SLE without LN, little is known on the underlying pathophysiological and
molecular mechanisms, and few interventional research data exist on increased CV risk directly
related to endothelial inflammation/activation entailed in LN.
With a bed-to-bench and back approach, in this review we aim to highlight the current knowledge
on CV risk associated to LN, pathophysiological mechanisms of related endothelial injury and

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 possible future target therapies. The first part of this review will focus on state of the art of clinical
Accepted Article
research on CV risk assessment in LN and SLE, highlighting current gaps in this field, in which
the inclusion of CVE among trials’ outcomes has still to be systematically implemented. It should
be noted that, while international task forces are focusing on specific guidelines for the
management of CV risk in SLE patients, more strict recommendations to guide primary and
secondary prophylaxis for CVE in patients with LN are still missing.
In the second part of this review, moving from bed to bench-side, we will first explore research
data on endothelial dysfunction in SLE and LN, and its connection with CV risk. We will then
discuss possible therapeutic perspectives specifically targeting molecular mechanisms underneath
endothelial dysfunction.

Cardiovascular Outcome in Lupus Nephritis Trials

In order to explore the inclusion of CV outcomes in LN Trials, we conducted a research using the
key word “Lupus Nephritis” in ClinicalTrial.gov registry, with 46 registered studies being active
by June 1st, 2020, 29 interventional and 17 observational studies. Systematic search was
performed blindly by two operators. If consensus was not achieved, a third opinion was requested.
Research strategy performed according to the PRISMA systems (a complete list of considered
studies is provided as Supplementary Material) focusing on the interventional trials, interestingly
none of the 29 retrieved studies on LN specifically takes into consideration CV as primary or
secondary outcomes.
CV events can be also indirectly assessed through score systems used to evaluate SLE activity.
The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), Safety of Estrogen in
Lupus National Assessment (SELENA)-SLEDAI and SLEDAI 2K are the most often adopted
scores and, at least one of those, was used in 14 out of the 29 (48%) mentioned trials. Of note,
these scales do not specifically categorize and characterize CV events, except for cerebrovascular
accidents. On the other hand, the British Isles Lupus Assessment Group (BILAG) Index, a more
comprehensive score system, which includes for example thromboembolism, stroke, cardiac
failure, and myocarditis, was used in 4 of the 29 studies. (13%) (NCT03580291, NCT02226341,
NCT02936375, NCT03917797). The Systemic Lupus International Collaborating
Clinics/American College of Rheumatology (SLICC/ACR) Damage Index, which represents the
most specific score to estimate CV consequences of the disease, enlisting coronary disease,
myocardial infarction, stroke, was used just in 2 of the retrieved trials (NCT03943147,

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 NCT03747159). Notably, 15 out of 29 (52%) studies did not use any of the above-mentioned
Accepted Article
scores. Moreover, all the included studies have a short term follow up, ranging from 6 months to 3
years (most frequently one year), which greatly limits the possibility to reach solid conclusions on
the association between CV disease and SLE.
When looking at the 17 observational studies, 5 were long-term national registries collecting a
wide range of data (NCT03001973, NCT00001372, NCT03929887, NCT01802034,
NCT00001979), and among them only 2 specifically included CV events as outcomes
(NCT03001973, NCT03929887). One of these repositories is specific for LN, one is dedicated to
SLE, while three consider autoimmune kidney diseases in general. Only one of the other 12
observational studies used SLEDAI score (NCT04218890), while all others did not clearly adopt
any scale of disease activity with information on CV events.
When widening our research on ClinicalTrial.gov registry using “Systemic Lupus Erythematosus”
as query, 170 active registered trials were found, 124 adjunctive to the previously retrieved
records. Among them, only 15 of these trials enrolled patients with LN, considered CV events as
clinical outcomes (directly or indirectly through BILAG or SLICC/ACR Damage indexes), and
had a follow up of at least one year. Three out of 15 were registries –one for rheumatologic
diseases (NCT04402086), and two specifically designed for SLE (NCT00001372 and
NCT04320680) evaluating a broad spectrum of clinical and laboratory data with a follow up of
five years or longer. A fourth registry from Mayo Clinic focused on spontaneous coronary artery
disease (CAD) in autoimmune diseases (NCT03941184). Eight out of 15 (53%) specifically
addressed drug therapies and evaluated CVE indirectly through BILAG or SLICC/ACR Damage
indexes. Of note, three out of 15 (20%), specifically addressed the problem of accelerated
atherosclerosis and increased CV risk in patients affected by SLE (NCT04276701, NCT04037293,
NCT01180361). Mentioned studies are summarised in Table 1.
Our analysis, albeit far from being conclusive, showed that despite the known tight link between
SLE and CV morbidity and mortality, evidences to guide interventional approaches in the
management of CV on the affected patients are still based on real word experience with a very
limited number of trials specifically evaluating this complex issue.
In order to contribute to the debate on this relevant topic, we herewith discuss the pathophysiology
of the endothelial dysfunction in LN setting, highlighting those aspects that might represent
potential interventional target.

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 Endothelial dysfunction in SLE and LN
Accepted Article
The concept of endothelial cells (ECs) being a mere internal pavement of blood vessel has long
been abandoned. It is now clear that endothelium is a fine regulator of tissue and blood
physiology. Therefore, reaching a better understanding of its role in pathophysiological processes
is of pivotal importance in order to develop therapeutic strategies for all those pathologies sharing
a detrimental vascular component, including SLE and LN. In patients affected by SLE, endothelial
injury leads to an increased risk for CV disease, primarily stroke, large calibre and peripheral
arteries disease, and venous thromboembolisms including deep vein thrombosis and pulmonary
embolism [11].To date, a number of biomarkers of endothelial dysfunction have been identified
and their association with CVE has been documented, including: Platelet Endothelial Cell
Adhesion Molecules (PECAM), Vascular Endothelial-cadherin (VE-cadherin) and -Growth Factor
Receptor (VEGFR-2), and specific tyrosine kinase receptor (Tie-2), to name a few [12–14]. Most
interestingly, the signalling cascades triggered by the above-mentioned factors, increase CV risk
and are found to be dysregulated in SLE and LN [15–18]. Cell adhesion molecules (CAMs)
expressed on ECs play an essential role in the inflammatory cascade mediating activation,
adhesion and migration of different subsets of leukocytes. PECAM-1 and several other adhesion
molecules, are significantly upregulated in SLE patients with a correlation between higher plasma
levels and severity of symptoms[15], while others studies report a colocalization of PECAM-1 and
interleukin (IL)-7 in kidneys of patients affected by glomerulonephritis [19]. Activated leukocyte
cell adhesion molecule (ALCAM) has recently been identified as a promising novel urinary
biomarker to detect the existence of a renal involvement in SLE patients and to discriminate active
from quiescent LN. ALCAM is a cell surface glycoprotein expressed by endothelial cells that
mediates T cell activation, adhesion and migration. Its enhanced expression in renal tissue was
detected in aMRL/lpr lupus-like glomerulonephritis mouse strains [20]. Two recent cross-sectional
studies found increased urinary levels of ALCAM in LN patients compared to SLE patients
withouth renal involvement and healthy controls [21,22]. Moreover, ALCAM levels were
significantly higher in patients with active LN, compared to quiescent LN [21,22] and, among
active LN, were higher in patients preseting prolipherative LN compared to membranous LN [21]
arthritis. ALCAM role in the development of LN remains to be further elucidated. However,
urinary levels of ALCAM may represent a simple and non-invasive tool to determin presence of
renal involvement, its phenotype, being higher in proliferative LN, and a marker of response to
therapy/renal disease activity, being lower in quiescent LN.

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 Analogously, VE-cadherin is linked to the development of CV disease and serum levels are
Accepted Article
increased in rheumatoid arthritis and SLE patients, with an hypothesised mediation of circulating
microparticles and associated immune complexes [23]. As for VEGFR-2, there are several reports
about its role in the onset and progression of autoimmune diseases, including SLE and LN [24].
In a mouse model of SLE, for instance, inhibition of VEGFR-2 was showed to aggravate kidney
disease and to increase mortality, but the underling pathogenic mechanisms remain to be
elucidated [25]. On the other hand, although it is unclear whether the augmented levels of
VEGFR-2 are associated with increased susceptibility to SLE, their role in SLE-related vascular
injury has been confirmed [26].
A major role in regulating ECs functions is played by endothelial Nitric Oxide Synthase (eNOS)
with a redox unbalance resulting from its compromised activity under pathological conditions,
leading to radicals production [27]. In SLE setting, in vivo eNOS expression is found significantly
reduced in samples from SLE patients and this mechanism has been postulated to be a mediator of
endothelial activation and NO-dependent pathological cascades [28].
In a murine model of SLE, eNOS was found to be inhibited through phosphorylation, while
superoxide production is increased in aortic endothelium, with an impaired vasodilatory reaction
to acetylcholine, a transversal benchmark for endothelium-dependent vasorelaxation [29]. In
another study performed on human umbilical vein endothelial cells from SLE patients and
matched controls, altered NOS activity and NO-modulated cascades were proved to be linked to
compromised ECs [30]. These findings are further supported by genetic studies on eNOS
polymorphisms as risk factors for SLE [31,32].
Another marker of possible relevance in SLE and related CV increased risk evaluation is Annexin
V. In fact, in a SLE cohort, Annexin V serum levels were found to be an independent predictor of
endothelial dysfunction and of subclinical atherosclerosis evaluated through the intima-medial
thickness (IMT). Annexin V could be considered as a biomarker of early atherosclerosis, still to be
validated [33].
The presence of antiphospholipid antibodies (aPL) is another well-described risk factor for
thrombosis [34] to include events affecting the renal vasculature [35]. aPL have been associated
with an increased cardiovascular risk, even in the young populations [3]. More recently, novel
antibodies specificities, namely anti-beta-2-glycoprotein I domain 1, have been shown to be
associated with aPL-related injuries in patients with concomitant LN, paving the way for more
accurate risk stratification strategies in the management of patients with SLE and aPL.

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 Contextually, available evidence suggest a role for anticoagulation in patients with concomitant
Accepted Article
LN and thrombotic microangiopathy, especially when aPL are detectable [36]. Future studies
specifically designed to assess the benefit of prolonged anti-thrombotic strategies to reduce the
overall CV burden, are needed.
Although some processes are known to be common for the entire vascular bed, it is clear that
differential pathways of endothelial activation are expressed at microcirculation level compared to
macrocirculation, as well as in the venous bed compared to precapillary/capillary sections. A
growing amount of evidences have proved the existence of significant differences between arterial
and venous ECs in terms of morphology, regeneration capacity, and biomolecular properties.
Other authors have suggested that arterial-venous shift of ECs may not only be driven by
hemodynamic parameters but, at least partially, by autonomic innervation determining specific
phenotypes [37]. Regardless of the different activation of transcription factors in arterial ECs
compared to venous ECs, different surface markers and related signalling cascades are primers of
endothelial activation/dysfunction in the vascular consequences of autoimmune diseases. In SLE,
the vascular manifestations show different features on the arterial side compared to the venous
compartment [34]. On the other hand, in LN the two components seems to act synergistically,
leading to renal damage and impaired kidney function [35]. In this context, the identification of
specific and differential pathways of activation of ECs between arterial and venous vasculature
could represent an effective tool in order to target treatment strategies, lowering CV risk and
potentially leading to a better outcome. A first specific marker to discriminate arterial from
venous ECs was described in 1998, showing how Ephrin-B2 is selectively produced by arterial
ECs whilst a member of its receptors, namely Ephrin-B4 (see Figure 1), can predominantly be
found in venous ECs establishing a crosstalk between the two [38]. In the following two decades,
Ephrin-B2 expression has been showed to be promoted by Notch receptors activation depending
on extracellular signal-regulated kinase (ERK) upregulation through VEGF signalling cascade in
arterial ECs [39–41]. However, there are no known correlations between Ephrin-B2 and renal
disease in the adult kidney, apart from a study reporting a disorganisation in the tubule structure
affecting its reabsorption capacity [42]. Most recently, the overexpression of Ephrin-B4 has been
demonstrated to lead to glomerulopathies in a transgenic murine model [43] and a putative role in
regulating the crosstalk between podocytes and ECs has been proposed [44].
In veins, several markers are reported for specification, including Chicken Ovalbumin Upstream
Promoter-transcription factor II (COUP-TFII, see figure 1) which is significantly over-expressed

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 in venous [45] and lymphatic ECs but not in arteries [46]. It has been reported that COUP-TFII,
Accepted Article
alternatively named NR2F2 (nuclear receptor subfamily 2-group F-member 2), is able to induce
the expression of Ephrin-B4 [45], to suppress differentiation toward an arterial phenotype through
the inhibition of Notch [47] and to downregulate plasminogen activator inhibitor type 1,
thrombospondin, and PDGF-β [48]. In addition to the embryonic role in determining cell fate,
COUP-TFII triggers other specific pathways, such as extracellular matrix (ECM) modulation,
lipid/metabolism, growth factors, junction proteins, inflammation and thrombosis [48], being
preferentially activated in adult venous compared to arterial ECs. Few studies, however, report an
involvement of COUP-TFII in the pathophysiological mechanisms leading to venous endothelial
dysfunction in autoimmune diseases [49]. On the contrary, in other pathologic conditions, venous
endothelial dysfunction has been demonstrated to play a crucial role in mediating vascular
damage. In this line, vein wall valvular incompetence seemed to be induced by an imbalance
between matrix-metallo-proteinases and their inhibitors leading to tissue remodelling and loss of
stiffness[50]. In addition, another example of SLE-related vascular disease mediated by
endothelial dysfunction is venous thrombosis, which is characterised by increased plasma levels of
von Willebrand factor (vWF) and soluble P-selectin and, in turn, pro-coagulation activities of ECs.
The thrombotic process involves monocyte adhesion through the release of pro-adhesive
molecules from ECs which is significantly higher in venous ECs [51]. Binding of monocytes to
ECs in veins, is increased by Tumour Necrosis Factor (TNF)-α and lipopolysaccharide (LPS),
whereas they do not increase the expression of VCAM-1 or E-selectin in arterial ECs [51].
Analogously, VE-cadherin expression is upregulated in venous ECs.
Finally, a role has been proposed forp38 mitogen-activated protein kinase (MAPK) being
activated through phosphorylation and leading to a pro-inflammatory status with increased
production of chemokines, especially IL-8 [52].

Pharmacological endothelial targets in SLE and LN

Considering what mentioned above, endothelial dysfunction is a key component to be considered
in order to define pharmacological approaches aimed to reduce risk for CV disease in SLE and
LN. One tempting perspective would be to replace the compromised endothelium with endothelial
progenitor cells (EPCs) to re-establish vascular wall integrity [53], especially in those patients
with increased CV risk factors and a related limited capacity of EPC production [54]. It is not well
defined, however, whether SLE patients have a diminished number, and functionally impaired

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 EPCs that are not able to effectively turn over the increased apoptotic ECs [55]. This limiting
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aspect of the EPC-based therapeutic approach needs to be explored in more robust protocols to
classify progenitor cells through surface markers [56]. To date, the limited efficacy of such
therapeutic approach needs indeed to be associated to pharmacological therapies that aim to
recover endothelial function targeting disrupted cellular mechanisms (Table 2).
Indeed, when focusing on available options to manage LN, the current immunosuppressive
therapy only partially meets both the ideal targets of keeping under control the diseases and, at the
same time, of having a protective effect on EC. While the most commonly used
immunosuppressive agents, such as cyclophosphamide, azathioprine, mycophenolate mofetil or
(more recently introduced in the field of LN) tacrolimus have shown their efficacy in managing
LN, they also cause endothelial dysfunction and vascular damage [57]. On the contrary, the use of
other medications, like hydroxychloroquine, are known to have a positive immunomodulator
effect on ECs [58,59]. However, hydroxychloroquine is not sufficient alone to manage patients
with LN.
More recently, the use of rituximab, an anti-CD20 chimeric monoclonal antibody, has been
investigated as part of the armamentarium to manage LN. Pérez-Sánchez and co-workers showed
early restoration of immune and vascular phenotypes in SLE patients after B-cell depletion. The
use of rituximab was associated to a re-balancing in the homeostatic equilibrium of the immune
system and endothelium [60].
All in all, it is undoubtful that, while several medications are available to induce remission in LN,
more specific target therapies are warranted to manage SLE and LN, including ECs dysfunction to
prevent and reduce the associated increased CV risk.
Adhesion molecules have been suggested as potential pharmacological targets in SLE-related
hypertension and promising data demonstrate how an agonist of peroxisome proliferator activator
receptor β/δ (PPARβ/δ) is able to mitigate vascular complications and renal injury in a murine
transgenic model (Table 2). These results are corroborated by evidences that the use of a selective
inhibitor of PPARβ/δleads to increased plasma levels of proinflammatory cytokines and to higher
degree in arterial hypertension severity, endothelial dysfunction, and organ injury [61]. On the
other hand, VE-cadherin phosphorylation can be limited by Sphingosine 1-Phosphate Receptor
1(S1P1) activation, which protect ECs from neutrophils-mediated vascular injury, preventing loss
of endothelium integrity [62]. Moreover, LN is characterized by an impaired ability of ECs to

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 cope with Neutrophils Extracellular Traps (NETs) activity and the consequent VE-cadherin
Accepted Article
degradation, which induce vascular leakage.
A subset of T-cells with immunosuppressive features, natural occurring T regulating cells
(nTregs), and induced Treg cells (iTregs), have been proposed as mediator of autoimmune
diseases, pointing out the existence of new potential pharmacological targets [63] with promising
results in animal models of SLE and LN [64]. Most interestingly, while nTregs are unstable under
pro-inflammatory conditions, iTregs better tolerate such environment and are more able than
nTregs to limit autoimmune response in this context [65]. The induction of CD8+ and CD103+
iTregs phenotype can be obtained in vitro through exposure to IL-10 and TGF-β [66] Re-infusion
of these cells showed significant recovery of renal function in a murine model of SLE/LN, with a
clear advantage when compared to CD103- cells, also revealing a potential role of TGF-β1 in the
mediation of impaired immune response [64]. In fact, suppression of B cells response by iTregs
has been shown to be mediated by TGF-β1 signaling cascade. Interestingly, a recent case-control
study on SLE patients with and without LN demonstrated that in the LN subgroup there was a
significant reduction in in serum levels of TGF-β1, compared to patients without LN [67]. The
same study reported also a clear inverse relation between severity of kidney injury and TGF-β1
levels [67]. Another study on a murine model of SLE demonstrated how low levels of TGF-β1 can
be reversed by administration of histone deacetylase-specific inhibitor(CKD-506), which also
prevented SLE-induced renal damage, with no adverse effects [68].Another specific inhibitor of
HDAC-6 (ACY-378) has been showed to prevent the decrease in serum levels of TGF-β1 during
SLE progression, accompanied by a marked reduction in the severity of glomerulonephritis with
reduced deposition of immune complexes [69]. These findings underline the role of TGF-β1 and
Tregs in proper regulation of the immune response, making it a possible target of therapy. For
example, Tregs response can be stimulated by administration of TGFβ and IL-2 encapsulated in T-
cell-targeted nanoparticles, as demonstrated in an in vivo SLE murine model, with very
encouraging results in terms of pathogenic suppression and renal preservation [70].
Most of the above-mentioned therapeutic targets aim to regulate the interaction between B and T-
cells, with an indirect effect of ECs. Another therapeutic strategy that deserves further
investigation in the future is targeting markers expressed on renal ECs and in renal tissue. For
example, the receptor tyrosine kinase Tyro3, Axl, Mer (TAM) and their ligands, Protein S and
growth arrest-specific 6 (Gas6), play a relevant role in the development of LN and could be
possible target of therapy. TAM and their ligands are involved in recognition and removal of

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 apoptotic cells by macrophages [71]. It was demonstrated that Mer is highly expressed on renal
Accepted Article
ECs [72], it promotes phagocytosis of apoptotic debris and inhibits cytokine production,
maintaining an immune homeostasis [73]. Mer-KO mice have impaired capability of properly
removing apoptotic cells and develop lupus-like autoimmunity and nephritis [73,74]. On the other
hand, Axl and its ligand Gas6 become upregulated only under inflammatory stimulation and
promote mesangial proliferation [73,74]. Serum levels of both soluble Axl and Mer are elevated in
LN patients and correlate with disease activity and severity, being higher in proliferative GN [75].
Mice KO for Axl or Gas6 are protected against development of nephritis [74,76].Inhibition of the
Axl/Gas6 pathway may represent a possible target of therapy. Interestingly, Protein S, vitamin K
dependent key factor in the coagulation cascade, is also ligand for Mer and promotes its activity in
inducing phagocytosis of apoptotic cells [77]. Moreover, free Protein S has been shown to
promote in vitro T cell mediated suppression of activated dendritic cells [78], and could represent
a possible adjunctive therapeutic tool in vivo.
Even few data exist on exploiting endothelial elements considering their different location, on
venous or arterial side As previously mentioned, EphB4 is expressed on venous ECs and may
mediate crosstalk between podocytes and ECs [43]. Its phosphorylation has been shown to
mitigate nephritis outcomes promoting glomerular repair, while its inhibition by NPV-BHG712
severely compromise the glomerular architecture with microaneurysm formation and podocyte
injury [44]. It is not clear to what extent venous ECs are involved in impairment of renal function.
Intriguingly, a venous EC membrane receptor, COUP-TFII, is involved is vessel architecture
disarrangement during angiogenesis [79]. Although mostly investigated for its role in cancer,
COUP-TFII is highly expressed in renal tissue [80] and a role in regulating response to
atherosclerosis in veins has been suggested [81]. This pathway and its role in vascular injury need
to be further elucidated and could represent another possible target of therapy.
Taken into account the different mechanisms involved, the management of CV risk in LN patients
should be based on multi-targeted approaches, including immunomodulation, anticoagulation or
endothelial repair strategies. The current challenge is to identify categories of risks of patients with
LN varying according to disease characteristics and activity. The simultaneous integration of
multi-omics approaches including but not limited to genomics, epigenomics, transcriptomics,
proteomics, and metabolomics is paving the way to more granular stratification risk assessment
strategies. Integrated approaches will lead on the one hand to a better understanding of the
mechanisms connecting identified genetic variation to key drivers and pathways and ultimately to

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 cardiovascular risk categories. On the other hand, integrated approaches will help in the
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characterization of novel drug targets
Conclusions.
CV disease remains the leading cause of morbidity and mortality in industrialised countries [82],
being often associated with co-morbidities of various aetiology. SLE, and especially LN, is
characterized by a significant increase in CV risk, due to the concomitant presence of both
traditional and disease-specific factors, spanning from a 2.8-fold increase risk of myocardial
infarction to an 8.5 fold-increase in LN patients [9]. Despite the magnitude of the problem, clear
and homogeneous guidelines specifically focused on CV disease prevention and management are
still lacking. In fact, interventional and observational clinical studies rarely focus on vascular
events and, when they do, most often are limited to cerebrovascular events. Indeed, some studies
take into consideration SLE-specific indexes that include CV outcomes such as coronary artery
disease, myocardial infarction, thromboembolism, stroke, heart failure and myocarditis.
Nevertheless, these studies suffer from short follow-up periods, which strongly limit possible
conclusions and observations (follow-up reaching a maximum of 3 years). On broader search, less
than 10% of the identified clinical trials for SLE considered indexes that specifically evaluate CV
risk.
The need for true translational research filling the gap between the clinal outcomes and pathogenic
mechanisms to guide future therapeutic strategy is high. Future researches are mandatory to
elucidate the role of markers of endothelial dysfunction leading to CV diseases in SLE and LN,
with a specific focus on differences between arterial and venous events. Some data are available
about the involvement of PECAM, ALCAM, VEGF-R2 and Tie-2 [12–14] but most of them rely
on in vitro experiments and /or animal models and need to be translated into clinical strategies.
Despite the evidence of a different activation of venous and arterial ECs in SLE and LN, limited
evidence is available to guide the design of potential therapeutic targets. In fact, modulation and
restoration of proper endothelial activity might lower CV risk in autoimmune patients.. This
approach is, however, impaired by profound translational gaps. Replacing ECs with EPCs is still
lacking support from basic research with no uniformed protocols available to identify and classify
such a promising strategy [56].
When focusing on available therapeutic tools, hydroxychloroquine and targeting B-cells have
shown some promising results on ECs dysfunction, being their use linked to restoration of immune
and vascular phenotypes in SLE. Moreover, the use of agonists of PPARβ/δ as well as S1P1 has

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 been proven to ameliorate renal outcomes, but these results are still irrelevant for clinical
Accepted Article
applications. More promising data are coming from iTregs, which are more stable than nTregs
under pro-inflammatory conditions and can mitigate the immune response in animal models [65]
Another therapeutic approach could be oriented ontargeting markers expressed on renal ECs and
in renal tissue. TAM receptor tyrosine kinases mediated pathways could help to maintain
phagocytosis of apoptotic debris, inhibit mesangial proliferation and dysregulation of the immune
response [62].
In conclusion, insights on cellular mechanisms of endothelial and vascular dysfunction could help
clinicians and researchers to understand the bases of autoimmune diseases and related CV risks
and outcomes, and to develop new therapeutic targets to mitigate the severe impact of CV
complications in SLE and LN patients.

Acknowledgement.
The authors have no conflict of interest to disclose. The study has been supported by “Fondo
Ex60%” and “Fondo Finanziamento delle Attivita Base di Ricerca”.
.

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 Tables and Figures
Accepted Article
Table 1.
Type of Outcome
Title Condition Study Type Location Identifier
Primary outcome
 CHU de
Bordeaux -
Proportion of patients
service de
who show
médecine interne
atherosclerotic
Bordeaux, France
plaque progression
Immune
defined by the  CHU de
Mediators and
absence of carotid Strasbourd -
Metabolites to service
plaque in patients at
Stratify Systemic d'Immunologie
baseline and its
Lupus subsequent Clinique
SLE Interventional NCT04276701
Erythematosus development at Strasbourg,

Patients at High follow-up evaluated France

Trials specifically focused on cardiovascular risk / events

Risk of by semi-automated  Universität

3D vascular Freiburg
Cardiovascular
ultrasound Freiburg,
Diseases
[ Time Frame: At Germany
baseline (Day 0) and  BiocrucesBizkaia
18 months from Health Research
baseline] Institute
Baracaldo, Spain
Quantitative change
in carotid intima
media thickness

Premature (CIMT) from

SLE baseline [ Time Peking Union Medical
Atherosclerosis in
Atherosclero Observational Frame: Year 5] College Hostipal NCT04037293
Systemic Lupus
sis Quantitative changes Beijing, Beijing, China
Erythematosus
in CIMT at 5-year
follow-up from
baseline will be
documented
Comparison of 27-
Atherosclerosis in
hydroxylase level in
Rheumatoid
THP-1 monocytes Winthrop University
Rheumatoid
Arthritis and incubated in human Hospital
Arthritis Observational NCT01180361
Lupus: Restoring plasma (RA patient, Mineola, New York,
SLE
Cholesterol SLE, normal control, United States

Balance psoriatic arthritis). [

Time Frame: 3 years]
High Quality Lupus Observational All-cause mortality The First Affiliated
NCT03001973
Evidence of Nephritis (registry) and cardiovascular Hospital,
R
e

This article is protected by copyright. All rights reserved

Accepted Article Chinese Lupus mortality of LN Sun Yat-sen University

Nephritis (HELP) patients [ Time Guangzhou,

Frame: 10years] Guangdong, China
A composite renal
outcome of LN
patients [ Time
Frame: 10years]
The composite renal
outcome defined as
doubling of serum
creatinine and ESRD
(defined as initiation
of dialysis or kidney
transplantation).
Death [ Time Frame:
60 months]
Documentation of
death from any cause
 KangWon
Deterioration of renal
National
outcomes [ Time
University
Frame: 60 months]
Hospital
Doubling of serum
Chuncheon,
creatinine compared
Korea, Republic
to baseline serum
of
creatinine
 Keimyung
30% decline in
University
KOrea Renal follow-up estimated
Dongsan Medical
BiobankNEtwoRk GFR (using the
Center
MDRD equation
System Lupus Daegu, Korea,
and/or the CKD-EPI
TOwardNExt- Nephritis Observational Republic of
equation) compared NCT03929887
generation (registry)  Chung-Ang
to baseline
Analysis Other GN measurement University
(KORNERSTON End stage renal Hosptial

E) disease defined as Seoul National

estimated GFR University

≤10cc/min, initiation Hospital

of maintenance SMG-SNU

dialysis or kidney Boramae Medical

transplantation. Center

Improvement of Severance

clinical outcomes [ Hospital

Time Frame: 60 Seoul, Korea,

months] Republic of

Remission of
glomerulonephritis
re

nt
es

di

and proteinuria
gi
st
ri

cl

rl

C
V
n
a

v
e

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Accepted Article <0.3g/day (patient
who have
proteinuria<0.3g/day
at baseline have no
improvement in
clinical outcomes)
Duke University
SLE
Duke Lupus Observational Medical Center
Cutaneous Not reported NCT00512694
Registry (registry) Durham, North
Lupus
Carolina, United States
Odds of autoimmune
disease in
Spontaneous
Coronary Artery
Dissection (SCAD)
cases compared to
controls [ Time
Spontaneous Frame: Through
SLE
Coronary Artery study completion, or Mayo Clinic
Other Observational
Dissection approximately 50 Rochester, Minnesota, NCT03941184
Autoimmune (registry)
(SCAD) and years (average age of United States
Diseases study participants)]
Autoimmunity
Incidence Rate of
SCAD [ Time Frame:
Through study
completion, or
approximately 50
years (average age of
study participants)]
Repository of
Novel Analytes Lupus
Leading to Nephritis Change in disease
The Rogosin Institute
Autoimmune, IgA GN Observational progression
New York, New York, NCT01802034
Inflammatory and CKD (registry) [ Time Frame: Up to
United States
Diabetic Diabetic 10 years]
Other Registries

Nephropathies Nephropathy
(RENAL AID)
National Institutes of
Immune System Lupus Health Clinical Center,
Observational
Related Nephritis Not reported 9000 Rockville Pike, NCT00001979
(registry)
Kidney Disease Other GN Bethesda, Maryland,
United States
National Institutes of
Study of Systemic Natural History of
Observational Health Clinical Center, 
Lupus SLE SLE [ Time Frame: NCT00001372
(registry) 9000 Rockville Pike,
Erythematosus 12/31/2050]
Bethesda, Maryland,

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Accepted Article United States
Correlate outcomes Yale New Haven
SLE
Rheumatology and biomarkers of Hospital
Other Observational
Patient Registry rheumatic diseases [ New Haven, NCT04402086
Rheumatic (registry)
and Biorepository Time Frame: 10 Connecticut, United
Diseases years] States
Systemic Lupus
Erythematosus Cohort lupus:
epidemiologic lupus
Within the Interventional University Hospital,
SLE study about diagnosis NCT04320680
Framework of the (registry) Brest, France
and follow up [ Time
Multidisciplinary
Frame: five years]
Consultation

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 Table 2. Summary of the main pharmacological targetsof SLE and LN and their effects.
Accepted Article Molecular mechanisms along with bibliographical references and experimental models are
reported.

Pharmacological target Maineffect Molecularmechanism Model Reference

EPCs re-endothelialization Re-establishment of vascular wall Carotid artery injury

↓ CXCR4/JAK 38
(Lacipidine) integrity nude mice

PPARP β/δ agonists (GW0742) Mitigation vascular complicacies Lupus (NZB/WF1)

- 42
and renal injury female mice

Prevention of loss of barrier

function; ↓ VE- HUVECs;
S1P1activators
cadherinphosphorylation S1P1 ECKO mice 43
Protection of ECs from neutrophils-
mediated vascular injury

CD8+/CD103+ iTregs autologous Significant recovery of renal

↑ TGF-β LN (cGVHD) mice 45
infusion function

Prevention of SLE-induced renal ↑ TGF-β;

Histone deacetylase inhibitors damage; Lupus (NZB/WF1)mice
↓ inflammatory cytokines
(CKD-506, ACY-378) 50, 51
Reduction of glomerulonephritis,
lower immunocomplex activation

Pathogenic suppression and renal

T-reg inductors ↑ TGF-β, ↑ IL-2 SLE TG mice 52
preservation

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Accepted Article
Figure 1. Description of the main surface markers of arterial and venous endothelial cells and
their principal role in ECs biology.

Figure 1. Description of the main surface markers of arterial and venous endothelial cells
and their principal role in ECs biology.
Notch1: promotes expression of EPH-B2 upon ERK activity and VEGF signalling cascade. It can
be inhibited by CUP-TFII and suppress differentiation to arterial EC;
NRP1(Neuropilin 1), NRP2 (Neuropilin 2): membrane glycoproteins regulated by VEGF,
involved in neoangiogenic processes and cell fate determination;
Eph-B2 (Ephrin type-B receptor 2), Eph-B4 (Ephrin type-B receptor 4): Eph-B2 is produced in
arteries while Eph-B4 is mainly present in veins. Overexpression of Eph-B4 is associated with
autoimmune response and glomerulopathies.
COUP-TFII (COUP Transcription Factor II): majorly activated in arterial ECs, is a pivotal
determinant of ECs fate;
Dll-4 (Delta like-4): gene related to expression of Notch ligands;

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 Hey1/2: Hairy/enhancer-of-split related with YRPW motif protein, two of the principal target gene
Accepted Article
for Notch, implicated in cell differentiation.

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