Sortilin drives hypertension by modulating sphingolipid/ceramide homeostasis and by triggering oxidative stress

Abstract

Sortilin is a glycoprotein mainly known for its role as a trafficking molecule directing proteins to specific secretory or endocytic compartments of the cell. Its actual contribution to essential hypertension has remained hitherto elusive. Combining top-notch in vivo, ex vivo, and in vitro approaches to clinical investigations, Di Pietro et al. explored the signaling pathway evoked by sortilin in endothelial cells and report on such exploration in this issue of the JCI. The researchers identified circulating sortilin as a biomarker associated with high blood pressure. Mechanistically, they demonstrate that sortilin altered sphingolipid/ceramide homeostasis, initiating a signaling cascade that, from sphingosine-1-phosphate (S1P), leads to the augmented production of reactive oxygen species. Herein, we discuss the main implications of these findings, and we anticipate some of the potential avenues of investigation prompted by this discovery, which could eventually lead to treatments for cardiometabolic disorders.

Full text

The Journal of Clinical Investigation
1
Sortilin drives hypertension by modulating
sphingolipid/ceramide homeostasis and by triggering
oxidative stress
Fahimeh Varzideh,1,2 Stanislovas S. Jankauskas,1,2 Urna Kansakar,1,2 Pasquale Mone,1,2 Jessica Gambardella,1,2 and Gaetano Santulli1,2
1Department of Medicine, Division of Cardiology, Wilf Family Cardiovascular Research Institute, Einstein–Mount Sinai Diabetes Research Center (ES-DRC), Fleischer Institute for Diabetes and Metabolism
(FIDAM), and 2Department of Molecular Pharmacology, Institute for Aging Research, Institute for Neuroimmunology and Inflammation (INI), Albert Einstein College of Medicine, New York, New York, USA.
What is sortilin?
Sortilin is an approximately  kDa sin-
gle-pass type I transmembrane glycopro-
tein and a member of the vacuolar protein
sorting  protein VPSP domain recep-
tor family, which also includes sortilin-relat-
ed receptor  SORL, also known as sorting
protein–related receptor containing LDLR
class A repeats SorLA and sortilin-relat-
ed VPS domain containing receptor 
sorCS . The gene encoding for
sortilin, SORT1 also known as neuroten-
sin receptor  NTR or glycoprotein 
Gp, resides on human chromosome
 at the band p. whereas it is located
on murine chromosome  and includes 
exons. The protein is composed of a short
cytoplasmic tail carboxyterminal domain,
involved in its internalization, a single
transmembrane helix, and a large extracel-
lular Vpsp domain  kDa making up
a cysteine-rich domain and a ten-bladed β
propeller with an inner tunnel that contains
multiple ligand-binding sites. Sortilin acts
as a receptor, a coreceptor, and a traficking
molecule that binds and directs proteins to
speciic secretory or endocytic compart-
ments of the cell and regulated pathways,
including lysosomes, endosomes, and exo-
somes, or to the cell surface, where it also
participates in the traficking of extracellu-
lar vesicles . Less than one-tenth of the
cellular sortilin pool is actually present at
the cell surface, whereas it is mainly locat-
ed in intracellular membrane structures
involved in the sorting of various ligands.
Sortilin and arterial
hypertension
Initially identiied in brain tissue and most-
ly studied in neurological disorders ,
sortilin has been later associated with a
number of pathological conditions, includ-
ing dyslipidemia, inflammation, insulin
resistance, atherosclerosis, and vascu-
lar calciication , . In this issue of the
JCI, Di Pietro and collaborators elegantly
demonstrate that sortilin is also function-
ally linked to endothelial dysfunction and
hypertension . The authors identiied
sortilin as a powerful biomarker associated
with high blood pressure, since the increase
in circulating sortilin levels alongside
sphingosine--phosphate SP and solu-
ble NADPH oxidase –derived NOX-
derived peptide was more pronounced in
uncontrolled hypertensive patients. This
trailblazing discovery has obvious implica-
tions for the stratiication and clinical man-
agement of hypertension.
Sortilin drives oxidative stress
and hypertension
Starting from the well-known contribution
of sortilin in lipid raft clustering and in
the intracellular traficking of acid sphin-
gomyelinase aSMase , an enzyme
responsible for the hydrolysis of sphingo-
myelin to ceramide and phosphocholine,
the authors surmised a functional role for
SP in the regulation of oxidative stress in
endothelial cells. Such a role was proven
through means of a combination of in vivo,
ex vivo, and in vitro models. Pharmacolog-
ic and genetic approaches revealed that SP
activated the type  SP receptor SP,
initiating a signaling pathway Figure 
that includes PKCε and proline-rich tyro-
sine kinase  PYK, also known as protein
tyrosine kinase  β PTKB, leading to
the generation of reactive oxygen species
ROS by NOX also known as cytochrome
B β chain, CYBB, or gp-phox. It is
Related Article: https://doi.org/10.1172/JCI146343
Authorship note: FV, SSJ, and UK are co–first authors.
Conflict of interest: The authors have declared that no conflict of interest exists.
Copyright: © 2022, Varzideh et al. This is an open access article published under the terms of the Creative Commons
Attribution 4.0 International License.
Reference information: J Clin Invest. 2022;132(3):e156624. https://doi.org/10.1172/JCI156624.
Sortilin is a glycoprotein mainly known for its role as a trafficking molecule
directing proteins to specific secretory or endocytic compartments of
the cell. Its actual contribution to essential hypertension has remained
hitherto elusive. Combining top-notch in vivo, ex vivo, and in vitro
approaches to clinical investigations, Di Pietro et al. explored the
signaling pathway evoked by sortilin in endothelial cells and report
on such exploration in this issue of the JCI. The researchers identified
circulating sortilin as a biomarker associated with high blood pressure.
Mechanistically, they demonstrate that sortilin altered sphingolipid/
ceramide homeostasis, initiating a signaling cascade that, from
sphingosine-1-phosphate (S1P), leads to the augmented production of
reactive oxygen species. Herein, we discuss the main implications of these
findings, and we anticipate some of the potential avenues of investigation
prompted by this discovery, which could eventually lead to treatments for
cardiometabolic disorders.

The Journal of Clinical Investigation
2J Clin Invest. 2022;132(3):e156624 https://doi.org/10.1172/JCI156624
thromboxane A and elevated proinflam-
matory and procoagulatory events as well
as ampliied permeability and increased
oxidative stress .
Production of nitric oxide NO,
obtained in endothelial cells from its precur-
sor -arginine  via the enzymatic action
of endothelial NO synthase eNOS, is
a hallmark of a properly functioning endo-
thelium. However, the exact molecular
and prostacyclin, has an atheroprotective
action dependent on antioxidant and anti-
inflammatory effects including inhibition
of leukocyte adhesion and migration, has
anticoagulant and proibrinolytic effects,
and counteracts vascular permeability. On
the flip side, a dysfunctional endothelium is
characterized by impaired vasodilation and
augmented vasoconstriction mainly medi-
ated by angiotensin II, endothelin-, and
interesting to note that both aSMase and
neutral sphingomyelinases nSMase are
redox-sensitive enzymes whose activities
are incremented by ROS, thereby creating
a harmful vicious cycle.
Endothelial dysfunction and
hypertension
A healthy endothelium promotes vasodila-
tion mostly attributable to NO, adenosine,
Figure 1. Main molecular pathways activated by sortilin and the sphingolipid/ceramide signaling cascade in the endothelial cell. Hypertensive patients
display endothelial dysfunction, dysregulation of the sphingolipid/ceramide homeostasis, and elevated oxidative stress. Sortilin levels are linked to endo-
thelial dysfunction and hypertension, and circulating sortilin is proposed as a biomarker associated with high blood pressure. Sortilin, by orchestrating the
intracellular trafficking of enzymes implicated in the metabolism of ceramides, signals through the S1P pathway, augmenting ROS production and eventu-
ally impairing endothelium-dependent relaxation. Specifically, S1P triggers S1P3-mediated signaling, leading to NOX2 activation via PKCɛ and PYK2. Sortilin
activated a precise pathway that is independent of specific eNOS phosphorylation sites, including Ser1177 and Thr494. The phosphorylation sites of eNOS can
activate or inhibit the enzyme; the inhibitory sites are Ser114 (phosphorylated by PKCε), Thr494 (phosphorylated by PKCε; actually, the threonine is in position
492 in human eNOS, in position 494 in murine eNOS, and in position 495 in bovine eNOS), Tyr657 (phosphorylated by PYK2); the sites activating eNOS are
Ser615 (phosphorylated by AKT and CaMKIV), Ser633 (phosphorylated by PKA, not included in the figure), and Ser1177 (phosphorylated by AKT and CaMKIV).
aCDase, acid ceramidase; CaMKIV, Ca2+/calmodulin-dependent protein kinase IV; DAG, diacylglycerol; DCS, dihydroceramide synthase (also known as ceramide
synthase 3 [CS3]); DES, dihydroceramide desaturase; ER, endoplasmic reticulum; Gq, G protein α subunit q; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol
1,4,5-trisphosphate receptor; IR, insulin receptor; KDHR, 3-ketodihydrosphingosine reductase; NOX2, NADPH oxidase 2; PI3K, phosphatidylinositol 4,5-bis-
phosphate 3-kinase; SPHK, sphingosine kinase; SPNS2, sphingolipid transporter 2; SPPase, S1P phosphatase; SPT, serine palmitoyltransferase.

The Journal of Clinical Investigation
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J Clin Invest. 2022;132(3):e156624 https://doi.org/10.1172/JCI156624
excluded. Indeed, in addition to its action at
the endothelial level, sortilin controls lipid
absorption from the intestine and regulates
glucose transporter type  GLUT storage
vesicles in skeletal muscle and adipocytes,
which is signiicant because a compromised
translocation of these vesicles is involved in
the development of diabetes; furthermore,
sortilin mRNA and protein are downreg-
ulated in adipose tissue and muscle from
obese ob/ob and db/db mice, and insu-
lin resistance has been shown to induce
hepatic sortilin degradation. Intriguingly,
in a murine model of diet-induced obesity
DIO, sortilin-deicient rodents exhibited
enhanced glucose uptake in insulin tol-
erance tests and gained less body weight
than wild-type mice . This favorable
metabolic effect could be at least in part
attributed to an altered aSMase activity,
most likely reducing the levels of ceramide,
which is a well-recognized negative modu-
lator of insulin signaling, by inhibiting the
insulin receptor substrate  IRS Figure
. Of note, SORL has also been shown to
promote insulin-induced suppression of
lipolysis in adipocytes by acting as a sort-
ing factor for the insulin receptor that redi-
rects internalized receptor molecules from
endosomes to the plasma membrane, thus
enhancing the expression of the insulin
receptor and strengthening insulin signal
reception in target cells .
At the level of pancreatic islets, sorti-
lin-derived peptides promote β cell surviv-
al via the CaMK/CREB signaling pathway
; similarly, when associated with a neu-
rotensin receptor, sortilin protects pancre-
atic β cells from stress-induced apoptosis.
Instead, when associated with the p neu-
rotrophin receptor pNTR, sortilin could
favor β cell death.
Future directions for sortilin
interactomics and beyond
The novel indings summarized above will
certainly open up broad research oppor-
tunities. Future endeavors could include
identifying the molecular mechanisms
leading to sortilin cleavage/shedding most
likely released by activated platelets and
determining whether changes in plasma
levels of sortilin may have causative or
consequential effects in arterial hyperten-
sion, studying the potential role of sortilin
in endothelial autophagy , which might
underlie the pathogenesis of hypertension
NO, whereas at higher concentrations
 μM, reached for instance in the
presence of thrombi, SP induces vasocon-
striction. Intriguingly, SPs are crucial in
SP-mediated vasoconstriction, as shown
in experiments conducted in cerebral arter-
ies from rodents ; thus, SPR signaling
in the endothelium could counteract SPR
vasoconstriction mediated by VSMCs.
On the other hand, ceramide has been
implicated in endothelial oxidative stress,
growth inhibition, alterations of the cyto-
skeleton, senescence, and apoptosis; none-
theless, its exact role in the regulation of vas-
cular tone remains debated. Despite some
reports that have shown that ceramide could
enhance eNOS expression and phosphor-
ylation, substantial evidence indicates that
ceramide is able to increase ROS produc-
tion both mitochondrial and cellular ROS,
reduce NO bioavailability, facilitate the
interaction of eNOS with its negative regula-
tor caveolin-, and potentiate the phosphor-
ylation of eNOS at negative regulatory sites.
To rule out alterations in eNOS phos-
phorylation in the signaling pathway acti-
vated by sortilin, Di Pietro and colleagues
cunningly measured eNOS phosphoryla-
tion at the activation site Ser as well
as at the inhibition site Thr, inding no
notable differences between human endo-
thelial cells stimulated with acetylcholine
alone and cells pretreated with sortilin
for one hour . However, other eNOS
phosphorylation sites were not explored,
including the inhibitory sites Ser, which
is targeted by PKCε, and Tyr, which is
targeted by PYK Figure . Remarkably,
increased phosphorylation at any of these
sites would not have contradicted the ind-
ings shown in the paper, inasmuch as both
PKCε and PYK are downstream of the sig-
naling pathway that follows SP activation
by SP, and PYK can activate NOX lead-
ing to increased ROS production and at
the same time is further activated by ROS
Figure . Moreover, Joel Karliner’s team
had previously shown in a murine model of
myocardial ischemic preconditioning that
sphingosine kinase is a target of PKCε .
Sortilin and insulin resistance
The il rouge linking endothelial dysfunc-
tion, hypertension, and diabetes is complex
and not fully understood, and a primary
role for sortilin in the common mecha-
nisms underlying these processes cannot be
mechanisms leading to reduced NO gen-
eration, as observed in hypertension, are
not fully clear. The experimental evidence
provided by Di Pietro et al.  combining
clinical and preclinical data adds another
key piece to the jigsaw puzzle of essential
hypertension.
Is there a vascular ceramide/
S1P rheostat?
For years, sphingolipids have been consid-
ered mere intermediate products of sphin-
gosine degradation until their complex
effects began to be delineated. Indeed, in
addition to being structural components of
the eukaryotic membranes, sphingolipids
act as signaling molecules that regulate a
number of biological functions. In , two
interconvertible sphingolipid metabolites,
namely, ceramide and SP, were identiied
as major determinants of cell fate, a concept
referred to as the “sphingolipid rheostat”
, attempting to connect several inde-
pendent indings evidencing the capacity of
ceramide and SP to differentially regulate
cell survival and cell growth by modulat-
ing opposed signaling pathways. Increased
ceramide levels induce cell growth arrest
and apoptosis, while SP suppresses cera-
mide-mediated apoptosis and is necessary
for cell proliferation induced by growth fac-
tors. Later on, ceramide and SP emerged as
fundamental regulators of vascular tone 
at the level of both endothelial and vascular
smooth muscle cells VSMCs.
SP can act as a paracrine or autocrine
mediator by binding to speciic membrane
receptors divided into ive subtypes: SPR–
 that orchestrate fundamental biologi-
cal processes, including cell proliferation,
migration, chemotaxis, cytoskeleton orga-
nization, angiogenesis, and mitogenesis.
These receptors are also involved in immune
modulation and in the suppression of innate
immune responses from T cells. Therefore,
the signaling pathways linking SP and ROS
generation could be implied in the two-way
relationship between cardiovascular and
immune systems, which might be involved
in the pathophysiology of several cardiomet-
abolic disorders, especially considering that
sortilin regulates the secretion of proinflam-
matory cytokines from immune cells .
S1P receptors and vascular tone
At nanomolar concentrations, SP elicits
endothelium-dependent vasodilatation via

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especially given the interaction between
sortilin and PCSK .
Based on recent transcriptomic analy-
ses in zebra ish showing that mutations in
SORL affect mitochondrial function ,
further studies are warranted to determine
whether sortilin could regulate mitochon-
drial itness and mitochondrial ROS produc-
tion. Last but not least, sortilin dimerization
has been implied in the formation of extra-
cellular vesicles, a heterogeneous group of
cell-derived membranous structures com-
prising exosomes and microvesicles Figure
, which regulate intercellular communica-
tion by transferring microRNAs, proteins,
and lipids to neighboring cells . Since
endothelial extracellular vesicles have
emerged as critical players in a number of
cardiovascular and cerebrovascular disor-
ders, including the systemic manifestations
of COVID , we anticipate that excit-
ing research will stem from the just-estab-
lished participation of sortilin in endotheli-
al dysfunction.
Author contributions
The order of co–irst authors was deter-
mined by drawing lots.
Acknowledgments
The Santulli lab is supported in part by the
NIH RHL, RDK,
RHL, RDK, R-
AG, THL, and R-
DK to GS, the Irma T. Hirschl and
Monique Weill-Caulier Trusts to GS, and
the Diabetes Action Research and Edu-
cation Foundation to GS. FV, SSJ and
JG hold postdoctoral fellowships from
the American Heart Association AHA-