2. cells, too (Ryglewski et al., 2007; Munaron and Fiorio Pla, 2009). There-
by, the endothelium-protective effects of lithium may be important in
cerebrovascular disease, traumatic brain injury with disturbed vascular
regulation (Rajkowska, 2000; Bosche et al., 2003; Dohmen and Bosche
et al., 2007; Gold et al., 2011; Halcomb et al., 2013; Leeds et al., 2014),
and even bipolar disorder (Goldstein and Young, 2013). In endothelial
cells, lithium prevents the discharge of calcium from endogenous stores
by inhibition of the inositol trisphosphate (IP3)-sensitive calcium chan-
nels of the endothelial endoplasmic reticulum (ER) (Schäfer et al.,
2001), thus, counteracting cells stress-induced calcium overload and con-
ferring lithium a cytoprotective potential (Bosche et al., 2013), possibly
through inhibition of GSK-3β (Rej et al., 2015b) and/or IMPase (Chiu
and Chuang, 2010; Dutta et al., 2014). Functionally, maintenance of intra-
cellular calcium homeostasis together with other lithium effects manifest
as modified endothelium-mediated vasodilation (Förstermann and
Münzel, 2006), a potential marker of preserved or improved endothelial
health (Yoo and Kim, 2009; Grove et al., 2015).
In long-term or at supratherapeutic levels, i.e. above the generally
recommended concentrations in humans (Rej et al., 2015a), lithium
can impair particularly kidney and brain function (Laliberté et al.,
2015), where it tends to accumulate (Lichtinger et al., 2013;
Johnson, 1998). Historically, lithium toxicity has been linked to in-
hibition of the GSK-3β or inositol monophosphate pathway leading
to disturbed cellular metabolism (Rybakowski et al., 2013;
Trepiccione and Christensen, 2010) On the other hand, lithium ac-
cumulation or toxicity may also be related to vascular hemodynam-
ic abnormalities (Schou et al., 1968; Laliberté et al., 2015). One
could postulate that the effect of lithium could be harmful to endo-
thelium at high doses and impairs vasodilation, which may contrib-
ute to tissue specific toxicity, e.g. in the brain and kidney (Lichtinger
et al., 2013; Johnson, 1998).
How can lithium be protective to blood vessel endothelium at low
therapeutic doses, but be harmful to blood vessels at higher or
supratherapeutic doses? We hypothesize that at high levels, the effect
of lithium on endothelium reverses — that lithium impairs endothelium-
dependent relaxation of blood vessels. To validate this hypothesis, an
established approach was applied to study vascular function in vessel
grafts (Ebner et al., 2011; Mulvany and Halpern, 1977; Kopaliani et al.,
2014), which recently modified and translated also for human use
(Wilbring et al., 2013).
Yet, to our knowledge, the data presented here uniquely suggested
that supratherapeutic and higher therapeutic, but not lower therapeutic
lithium levels impair particularly endothelium-dependent vascular re-
laxation underlining the concept of opposite effects of lithium at differ-
ent therapeutic concentrations.
2. Material and methods
2.1. Ethics of the animal model
This experimental study was approved by the University Commis-
sion on Animal Experiments with respect to the animal welfare regula-
tions of Germany, in accordance to the European Communities Council
Directive and to the National Institutes of Health (NIH) Guidelines. For
performing the study, written permission was obtained from local
authorities.
2.2. Materials and drugs
All materials, reagents and drugs are described when mentioned
within their respective method sub-sections (see below).
2.3. Cold storage solutions
The used cold storage Tiprotec™ solution (Dr. F. Köhler GmbH,
Bensheim, Germany) contains the following substance concentrations
(all are given in mmol/L): α-ketoglutarate 2, aspartate 5, N-acetyl-histi-
dine 30, glycine 10, alanine 5, tryptophan 2, sucrose 20, glucose 10, Cl−
:
103.1, H2PO4: 1, Na+
: 16, K+
: 93, Mg2+
: 8, Ca2+
: 0.05, deferoxamine:
0.082 and LK 614: 0.017. The pH (at 20 °C) was 7.0 and the osmolarity
was 305 mosmol/L. This standard solution served as the control precon-
ditioning or was supplemented with the different lithium chloride
(LiCl) or lithium acetate (LiAc) concentrations.
2.4. Murine and porcine vessel preparation
The vessel grafts were isolated from murine aortas or from porcine
middle cerebral arteries (MCA). Vessel preparation was performed ac-
cording to the slightly modified method for rodents as described in de-
tail elsewhere (Ebner et al., 2011; Wilbring et al., 2013; Kopaliani et al.,
2014). In brief, male CD57 mice 8 to 10 weeks of age (Charles River Lab-
oratories, Sulzfeld, Germany) were sacrificed by cutting off the upper
cervical spinal cord under deep anesthesia. Male swine (Sus scrofa
domesticus, 24 to 26 weeks of age) were stunned by electroshock and
sacrificed by exsanguination. All mice and swine were dissected imme-
diately. The murine aortas (pars thoracalis without aortic arch) or the
proximal part (M1 segment) of porcine middle cerebral arteries were
recovered and directly placed into a storage solution containing either
1) Tiprotec™ solution only (Dr F. Köhler GmbH, Bensheim, Germany)
or 2) modified Tiprotec™ solution supplemented with 0.4 to
100 mmol/L lithium (LiCl/LiAc, Sigma-Aldrich, Taufkirchen, Germany)
and stored at 4 °C for ≥48 hours (h).
2.5. Post-mortem model of vascular tone control
The post-mortem model with de-nerved vessel grafts was per-
formed to investigate the isolated vessel reaction in response to differ-
ent lithium concentrations independently of the influence of lithium
on the central and thereby also on the vegetative nerve system includ-
ing its remote control of the vessel tone.
2.6. Type of vessel
The thoracic aorta (pars descendens) was chosen as the used vessel
type, because a) it is an elastic type artery containing both the ordinary
vascular smooth muscle cells (SMC) and the myointimal SMC in a rela-
tively high number; moreover, because b) aortic endothelial cells were
used in our previous vessel graft and cell culture studies regarding cyto-
solic [Ca2+
] measurements after long-term and immediate use of lithi-
um and its influence on the specific type of endothelial cells taken
from the aorta (Schäfer et al., 2001; Bosche et al., 2013). Vessel grafts
of the MCA (M1 segment) from porcine brains were additionally used
to investigate the influence of lithium on cerebral endothelium-depen-
dent vasorelaxation.
2.7. Isometric force measurement
After cold storage aortic or cerebral vessel grafts (pipes or long rings,
2 mm in length, 500–600 μm or 1150–1400 μm internal width, respec-
tively) were transferred to a 90%/10% (vol/vol) mixture of phosphate-
buffered saline (PBS) solution and Hank's balanced salt solution
(HBSS, Sigma-Aldrich, Taufkirchen, Germany) gently warmed to
37 °C/98.6 °F over 30 min. Then, the vessel grafts were studied,
stretched with a resting tension equivalent to that obtained by exposure
to an intraluminal pressure of 20 mm Hg for maximal responses. Vessel
rings were equilibrated for 10 min prior to vasomotor analysis. Maximal
contraction was induced by exposure of vessel rings to a potassium-
enriched solution (123.7 mmol/L KCl) and/or to the application of
10 μmol/L phenylephrine (see below Vasoactive Agents). Vessel relaxa-
tion toward acetylcholine (ACH; 10−8.5
to 10−5.5
mol/L) or sodium ni-
troprusside (SNP; 10−8.5
to 10−5.5
mol/L) was tested after a plateau
constriction induced by 10 μmol/L phenylephrine to assess
99B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106
3. endothelium-dependent and endothelium-independent relaxations in
aortic vessel grafts, respectively. Since middle cerebral arteries do not
constrict in response to the α1-adrenoceptor agonist phenylephrine
the vessel grafts were studied under native conditions at resting tension
equivalent to an intraluminal pressure of 20 mm Hg. For the analyses of
the influence of the different lithium preconditions, we focused on the
following ACH and/or SNP additions: a) none, b) 10−8.5
mol/L,
c) 10−7
mol/L) causing approximately half and d) 10−5.5
mol/L causing
the most pronounced vessel relaxation found in pilot experiments.
We measured vessels' isometric forces to constrict and relax using a
commercially available myograph (DMT-610M, Power Laboratory/400;
AD-Instruments, Spechbach, Germany). The experiments were conduct-
ed according to the well-established and slightly modified method using
a Halpern–Mulvany-myograph (Mulvany and Halpern, 1977; Ebner
et al., 2011; Wilbring et al., 2013; Kopaliani et al., 2014). For data acqui-
sition, the tissue bath system 700MO™ was used with a PowerLab Data
Acquisition System™; and data recording was performed with
LabChart™ software (AD-Instruments, Spechbach, Germany).
2.8. Vasoactive agents
Phenylephrine (Sigma-Aldrich) was used to induce smooth
muscle cell (SMC)-mediated vasoconstriction. Acetylcholine
(Sigma-Aldrich) was used to stimulate the endothelial NO produc-
tion and thereby provoke endothelium-dependent vasodilatation.
Sodium nitroprusside (Sigma-Aldrich) was applied to induce endo-
thelium-independent vasodilatation by directly decreasing the vas-
cular SMC tone.
2.9. Inhibition of nitric oxide synthase (NOS)
In some experiments, L-NG
-monomethyl arginine (L-NMMA;
Sigma-Aldrich, Germany), a potent inhibitor of the endothelial nitric
oxide synthase (NOS) was used to inhibit the NOS before adding the va-
soactive drugs. In brief, we used L-NMMA in a concentration of
300 μmol/L to block the endothelium-dependent relaxation of the ves-
sel grafts, aiming to proof whether this specific type of vessel relaxation
is NOS mediated or independent of endothelial NOS activity.
2.10. Vessel denudation (removal of endothelium)
In another bench of experiments, the endothelium was denudated
from the vessel grafts according to the method previous described
(Wilbring et al., 2013; Kopaliani et al., 2014). In short, denudation of
the endothelium is a method to carefully remove endothelium from
the basal membrane of vessels. We somewhat modified the method
using a suture string for removing the endothelium. Denudation of the
endothelium aimed to totally block the endothelium-dependent part
of the vessel relaxation, or to independently investigate the vessel be-
havior without endothelium.
2.11. Inhibition of IP3-sensitive Ca2+
-release channel of the endothelial
endoplasmic reticulum
For blocking of the IP3 sensitive Ca2+
release channel of the ER
(Schäfer et al., 2001) 3 μM Xestospongin D (XeD, Calbiochem —
Merck Millipore, Darmstadt, Germany) was added to the vessel
grafts. Immediately and 30 min after XeD addition ACH and SNP in-
duced vessel relaxation was myographically measured after maxi-
mal contraction.
2.12. Statistical analyses
Normality and variance equality was assessed using Kolmogorov–
Smirnov- and Levene-tests. Normal distribution was found in all vari-
ables and groups. Results are expressed as mean ± SE. Intergroup
differences were analyzed using one-way analysis of variance with
post-hoc multiple comparisons, or independent-sample t-test. p b 0.05
was chosen as level of significance. Data analyses were performed
using GraphPad Prism 6 and IBM SPSS (IBM, Chicago, IL, USA).
Fig. 1. Effect of lithium-preconditioning at supratherapeutic concentrations on endothelium-
dependent and endothelium-independent vasorelaxation. Vessel graft of murine aortas were
preconditioned with Tiprotec™ solution alone as control (A), or Tiprotec™ supplemented
with 20 mM LiCl (B) or 100 mM LiCl (C) for 48 h. Afterwards the vessels were
precontracted by phenylephrine (10 μmol/L) and the capacity of endothelium-dependent
and endothelium-independent vasorelaxation was tested by additions of equal amounts of
10−8.5
, 10−7
or 10−5.5
M acetylcholine (ACH, red record line) or sodium nitroprusside
(SNP, blue record line), respectively. Representative recordings of isometric force
measurements of n = 7 per group are given. Horizontal bars indicate recording time of
2 min, vertical bars force development of 1 mN, horizontal bars 2 min, respectively.
100 B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106
4. 3. Results
3.1. Influence of lithium on endothelium-dependent vs.
endothelium-independent vessel relaxation
The influence of lithium on two modes of vascular response, the en-
dothelium-dependent and endothelium-independent relaxation, was
studied in vessel grafts exposed to ACH or SNP at concentration given
in Fig. 1A–C. In vessel grafts preconditioned with 20 or 100 mmol/L
LiCl for 48 h (Fig. 1B and C), ACH-induced vasorelaxation became re-
duced, whereas the effect of SNP on the vascular tone was left unaltered,
indicating that lithium affects the endothelium-dependent but not the
endothelium-independent vascular response. In line with that, ACH as
well as SNP led to a profound equivalent relaxation in non-
preconditioned vessel grafts (Fig. 1A).
In vessel grafts preconditioned with 20 mmol/L LiCl the direct com-
parison of ACH- vs. SNP-induced vasorelaxation (Fig. 2) revealed a sig-
nificant difference between the endothelium-dependent and -
independent vasorelaxation at all ACH and SNP concentrations tested.
Preconditioning with LiCl concentrations N20 mmol/L enlarged the sig-
nificant difference between both modes of vasorelaxation (data not
shown) reaching its maximum at a concentration of 100 mmol/L LiCl
(Fig. 3A). In contrast, preconditioning at that high LiCl concentration
did not affect SNP-induced reduction of the vascular tone (Fig. 3B), indi-
cating that the discrepancy between ACH- and SNP-induced vasorelax-
ation in lithium preconditioned vessel grafts is due to a loss of
endothelium-mediated vascular function.
3.2. Effects of lithium on vascular function seem to involve IP3-sensitive
calcium discharge of the ER and NOS activity in endothelium
To prove the concept that LiCl affects vascular function by targeting
the endothelial cells of the intima but not cells of the media, vessels
were denudated from endothelial cells by a classical mechanical ma-
neuver. After denudation, ACH failed to induce vasorelaxation in LiCl-
preconditioned as well as in non-preconditioned vessel grafts
(Fig. 3C). In contrast, denudation did not change the SNP-induced vessel
relaxation at all (Fig. 3D), suggesting that lithium precondition targets
endothelium-dependent but not endothelium-independent vascular
function. In line with that, L-NG
-monomethyl arginine (L-NMMA,
300 μmol/L), a potent inhibitor of the endothelial nitric oxide synthase
(NOS) and, therefore, of endothelium-dependent vasorelaxation,
abolished the relaxation induced by 10−5.5
mol/L ACH of both lithium-
preconditioned and non-preconditioned vessel grafts (Fig. 3E). Whereas
L-NMMA did not affect endothelium-independent vessel relaxation in-
duced by an equivalent concentration of SNP (10−5.5
mol/L) (107.9 ±
7.2% vs. 109.3 ± 6.1%, n = 7 per group, not significantly different).
Xestospongin D (XeD) is a reversible inhibitor of the IP3-sensitive
Ca2+
-release channel of the ER. Exposure to 3 μmol/L XeD for 30 min
did not affect the vascular tone of the phenylephrine precontracted ves-
sel grafts. However, it totally blocked the ACH-induced vasorelaxation,
but not the SNP-induced one (Fig. 3F). The similar responds to XeD or
LiCl preconditioning suggested that the impact of lithium on endotheli-
um-mediated vasorelaxation is also reliant on inhibition of the IP3-sen-
sitive Ca2+
-release of the ER.
3.3. Preconditioning with supratherapeutic lithium levels causes a
concentration-dependent reduction of endothelium-dependent
vasorelaxation
In a set of experiments, vessel grafts were preconditioned with
supratherapeutic LiCl concentrations of 10, 30 and 50 mmol/L. Subse-
quently, the vessels were precontracted with phenylephrine and the ca-
pacity of vasorelaxation in response to ACH was determined. As shown
in Fig. 4A the most pronounced differences in vasorelaxation were ob-
served at an ACH concentration of 10−7
mol/L. At that concentration,
a trend to reduced endothelium-dependent vasorelaxation was already
found in vessel grafts preconditioned at lower supratherapeutic LiCl
concentrations (10 mmol/L). At higher LiCl concentrations (30 and
50 mmol/L) ACH-induced vasorelaxation was reduced in concentra-
tion-dependent manner (Fig. 4A). To rule out a chloride ion effect, lith-
ium acetate was used for preconditioning. A similar concentration-
dependent reduction of endothelium-dependent vasorelaxation was
found in preconditioned vessel grafts with 20 and 100 mmol/L LiAc
(Fig. 4B).
3.4. Opposing effects of lithium at low vs. high therapeutic levels on
endothelium-dependent vessel relaxation
Preconditioning of vessel grafts with LiCl at a therapeutic concentra-
tion of 0.4 mmol/L slightly enhanced endothelium-dependent vasore-
laxation compared to non-preconditioned controls (Fig. 5). However,
preconditioning of the vessel grafts with LiCl at higher, but still thera-
peutic concentration (0.8 mM), reduced the endothelium-dependent
vasorelaxation, which had already been observed at supratherapeutic
lithium concentrations; though, the opposing effect of 0.4 mmol/L
Fig. 2. Comparisons of endothelium-dependent and endothelium-independent relaxation of murine vessel grafts preconditions at supratherapeutic lithium chloride concentration
(20 mM). Vessels were precontracted by phenylephrine (10 μmol/L) and vasorelaxation capacity was tested by acetylcholine (ACH) vs. sodium nitroprusside (SNP) added in
equivalent concentrations of (A) 10−8.5
, (B) 10−7
or (C) 10−5.5
mol/L, respectively. Data of n = 7 independent vessel grafts per group are given, *p b 0.05.
101B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106
5. 102 B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106
6. lithium was significantly higher compared to 0.8 mmol/L lithium chlo-
ride. Repeating these experiments using 0.4 and 0.8 mmol/L LiAc con-
firmed the results of LiCl (at 10−7
M ACH, n = 4, p b 0.05, 0.4 vs.
0.8 mmol/L LiAc). The effects were similar across all higher therapeutic
and supratherapeutic concentrations regardless of lithium salt, indicat-
ing that the opposing effects on vascular function solely relies on the ac-
tual concentration of lithium ions.
To test whether this opposing effect of lithium also occurs in the ce-
rebral vascular provinces, proximal M1 segments of middle cerebral ar-
teries, preconditioned at 0 (control), 0.4 and 0.8 mmol/L LiAc, were
challenged by 10−7
M ACH. As shown in Fig. 6, preconditioning of cere-
bral arteries with 0.4 mmol/L significantly enhanced the ACH-induced
vasorelaxation, whereas vessel grafts preconditioned with 0.8 mmol/L
LiAc significantly diminished the ACH-induced vasorelaxation. Compar-
ing 0.4 vs. 0.8 mmol/L LiAc preconditioning also revealed a significantly
different endothelium-dependent vasorelaxation. An increase of the
LiAc concentration and prolonged preconditioning (N48 h) led to fur-
ther impairment of the endothelium-dependent vasorelaxation; pre-
conditioning with 50 mmol/L LiAc, entirely diminished the ACH-
induced (10−7
M) vasorelaxation. Taken together, the results shown
in Figs. 5 and 6 suggest that lithium preconditioning induces opposing
effects on endothelium-dependent vasorelaxation and that this differ-
ential impact of lithium on endothelial function is a systemic phenome-
non not restricted to single vascular provinces.
4. Discussion
The present study is the first to demonstrate that high therapeutic
and supratherapeutic lithium levels impair endothelial function by
interfering with endothelium-dependent (but not endothelium-inde-
pendent) relaxation of blood vessels. This lithium effect moreover inter-
fered with endothelial integrity (denudation), NOS function and IP3-
sensitive calcium release of the ER, and was found in two different
Fig. 3. Impact of supratherapeutic lithium concentrations on endothelial function. Comparison of endothelium-dependent and independent vasorelaxaton in lithium-preconditioned (LiCl;
100 mmol/L) or non-preconditioned (control) murine vessel grafts. (A) ACH (10−5.5
mol/L) or (B) SNP (10−5.5
mol/L) were add to phenylephrine (10 μmol/L) precontracted vessels and
vasorelaxation were determined after 1 min. (C) and (D) comparisons under identical experimental conditions as in (A) and (B), however, after denudation of the endothelium from the
vessel grafts. Dashed lines represent the controls in (A) and (B) without denudation, respectively. (E) Comparison under identical experimental conditions as in (A), but in presence of L-
NG
-monomethyl arginine (L-NMMA, 300 μmol/L), a pan-specific inhibitor of nitric oxide synthase. Dashed line represents the control without L-NMMA as in (A). Data of n = 7
independent vessel grafts per group are given, *p b 0.05 compared to control. Please note that controls vs. LiCl in (C) and (D) and non-L-NMMA treaded control vs. LiCl in (E) were not
significantly different, respectively. (F) Comparison ACH- and SNP-induced (both 10−5.5
mol/L) vessel relaxation in phenylephrine precontracted vessels after 30 min of Xestospongin
D (3 μmol/L), representative experiment, vertical bar 1.5 mN, horizontal bar 2 min.
Fig. 4. Effect of lithium preconditioning at supratherapeutic concentrations on
endothelium-dependent vasorelaxation. (A) Murine vessel grafts were preconditioned
at 10, 30 and 50 mmol/L lithium chloride for 48 h. Afterwards the vessels were
precontracted by phenylephrine (10 μmol/L) and vasorelaxation capacity was tested by
acetylcholine (ACH). Data of n = 10–11 vessel grafts of independent preparations are
given. (B) Vessel grafts were treated as described in (A), however, preconditioned with
20 or 100 mmol/L lithium acetate. Data of n = 4 vessel grafts of independent
preparations are given, *p b 0.05 compared to control.
Fig. 5. Effect of lithium preconditioning at therapeutic concentrations on endothelium-
dependent vasorelaxation. Murine vessel grafts were preconditions at 0.4 or 0.8 mmol/L
lithium chloride (LiCl) for 48 h. Afterwards the vessels were precontracted by
phenylephrine (10 μmol/L) and vasorelaxation capacity was tested by acetylcholine
(ACH). Data of n = 3–4 vessel grafts of independent preparations are given, #
p b 0.05,
0.4 vs. 0.8 mmol/L LiCl.
103B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106
7. vascular provinces, moreover independent of the lithium salt. We
showed significantly different vessel relaxation responses to ACH after
0.4 and ≥0.8 mmol/L lithium preconditions. Our findings have major
clinical implications as well as for future research. Firstly, impaired en-
dothelium-dependent blood vessel relaxation may be a mechanism
contributing to acute lithium toxicity. Impaired endothelial function
and reduced vessel relaxation can have significant deleterious effects,
particularly in highly vascularized organs such as the brain and the kid-
neys that both control blood flow by vascular autoregulation. Even brief
exposure to lithium at high therapeutic or supratherapeutic levels as
low as N0.8–1.0 mmol/L for b12–24 h can have dramatic clinical effects
on renal and neurological function (Kessing et al., 2008; Kirkham et al.,
2014; Laliberté et al., 2015; Behl et al., 2015). One of the first pioneers in
lithium research, Mogens Schou had hypothesized that hemodynamic
abnormalities precipitated lithium-associated acute kidney injury
(Schou et al., 1968), although this has yet to be definitively validated.
In this respect, our current study may lead to better understand organ
specific effects of lithium on endothelial cells like in kidneys or brain,
as well as in specific vascular province, in which lithium may interfere
with systemic control mechanisms, the action of local vascular media-
tors or the (cerebro)vascular autoregulation of blood flow (Bosche
et al., 2010; Dohmen and Bosche et al., 2007).
Perhaps most importantly, our findings suggest that the intracellular
effects of lithium on the endothelium may be completely opposite
under supratherapeutic vs. therapeutic lithium levels. Since the inhibi-
tion of IP3 and GSK-3β molecular pathways have been implicated in
the usual functioning of lithium, one could hypothesize that lithium
causes hyper-functioning in those pathways (Berridge, 1993, 2014; Li
et al., 2012). This mechanism deserves more detailed investigations. In
addition to supratherapeutic lithium levels, we found that XeD, a specif-
ic inhibitor of the IP3-sensitive Ca2+
release channel of the ER,
completely blocked endothelium-dependent relaxation. This suggests
that supratherapeutic lithium levels likewise inhibit vessel relaxation
by interfering with the IP3-sensitive Ca2+
release from the ER. Under
physiologic conditions, Ca2+
released via this mechanism activates en-
zymes like nitric oxide synthases eliciting endothelium-mediated
vascular relaxation. Accordingly, blockage of this pathway immediately
impaired endothelium-dependent vascular relaxation similar to the
treatment with supratherapeutic lithium levels for 48 h. However, pre-
vious studies using therapeutic lithium levels have found that inhibition
of GSK-3β or IP3 pathways have been associated with improved vessel
relaxation (Dehpour et al., 2000; Riadh et al., 2011), such as shown
here for 0.4 mmol/L lithium. These animal studies used therapeutic
lithium levels in vivo over weeks or months, and had found somewhat
different results. However, most parts of the findings are consistent
with the protective effects we also observed when using two-day
0.4 mmol/L lithium exposure. On the other hand, different methods
and species may explain the minor conflicting parts of the results
(Dehpour et al., 2000; Riadh et al., 2011). It is possible that the known
endothelium protective effect of lithium is reversed at higher lithium
concentrations. This is supported by our data showing significant differ-
ences of vessel relaxation responses to ACH after 0.4 and 0.8 mmol/L LiCl
and LiAc precondition in two different species and vascular provinces. It
cannot be fully excluded, however, that other lithium-related mecha-
nisms are also involved (Grünfeld and Rossier, 2009; Chiu and
Chuang, 2010, 2012). Regarding the vascular field, the results of the
present study that lithium preconditioning can maintain or even im-
prove vascular function may help to understand lithium effects ob-
served in a variety of other clinical settings. Classic or remote ischemic
(pre)conditioning is an established concept to protect the kidney, heart
and brain with endothelium-dependent vascular function being one of
the important targets (González Arbeláez et al., 2013; Mergenthaler
et al., 2013; Liu and Gong, 2015; Wang et al., 2015). This concept is sup-
ported by recent studies showing that lithium-associated precondition-
ing reduces the risk of stroke in bipolar disorder patients (Lan et al.,
2015) and may support functional recovery after cortical stroke
(Mohammadianinejad et al., 2014). Interestingly, studies on the molec-
ular mechanism revealed that ischemic and lithium-induced pharmaco-
logical preconditioning share final common pathways involving
signaling elements like GSK-3β inhibition (Chiu and Chuang, 2010;
Talab et al., 2012; González Arbeláez et al., 2013). Whether both maneu-
vers of preconditioning may have synergistic effects remains an open
question, yet. Nevertheless, in the light of recent clinical and experimen-
tal evidence, our novel results can help to better understand the
underlining mechanisms of those remarkable clinical findings by mov-
ing the lithium-endothelium interactions into focus. Future investiga-
tions of the effects of therapeutic (and supratherapeutic) lithium
levels on endothelial function and vascular tone may yield great insights
into intracellular effects of lithium throughout the vasculature and
thereby the entire body.
Limitations of our study should be considered. We analyzed non-
perfused, cold storage, vessel grafts. Cold-storage and missing perfusion
might cause tissue bradytrophia and limited the time window for lithi-
um exposure and cellular up-take. Thus, relatively higher lithium con-
centrations within the solutions could lead to somewhat lower
cellular levels and less toxic effects compared to in vivo conditions
explaining our use of relatively high lithium concentrations in some ex-
periments. Similar methods and approaches to ours have also been used
by others (Ebner et al., 2011; Wilbring et al., 2013; Dutta et al., 2014).
Lithium sometimes takes two weeks to produce therapeutic effects in
bipolar disorder (Geddes and Miklowitz, 2013; Calkin and Alda, 2012).
According to the central nerve system and the vasculature, however,
lithium effects do not share same time courses. Still, acute lithium
toxicity often manifests in less than 24 h particularly at levels
N1.5 mmol/L both in humans and animals (Laliberté et al., 2015), sug-
gesting that we can be confident of our findings with higher therapeutic
and supratherapeutic lithium levels also in potentially bradytrophic
vessels. On the other hand, this argument could partly explain the
non-significant response at low therapeutic lithium levels such as
0.4 mmol/L compared to control, found in aortic vessel grafts which
can barely represent all vascular provinces. This experiment could also
be somewhat underpowered. Future studies in animals and humans
Fig. 6. Effect of lithium preconditioning at therapeutic concentrations on cerebral
endothelium-dependent vasorelaxation. Porcine brain vessel grafts (isolated from the
M1 segment of middle cerebral arteries) were preconditions at zero (control), 0.4 or
0.8 mmol/L lithium acetate (LiAc) for minimum 48 h. Afterwards vasorelaxation
capacity of native vessels (resting tension 20 mm Hg) was tested by acetylcholine (ACH,
10−7
M). Data of n = 7–8 vessel grafts of independent preparations are given in percent
(%) in relation to the ACH-induced vasorelaxation at control conditions (0 mM LiAc/
10−7
M ACH, 100%); *p b 0.01, LiAc 0.4 or LiAc 0.8 mmol/L vs. control, respectively;
#
p b 0.001, 0.4 vs. 0.8 mmol/L LiAc.
104 B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106
8. will need to assess whether longer—term therapeutic dosing of lithium
(e.g. 0.4–1.0 mmol/L) may be protective to endothelium, further clarify
the underlying molecular mechanisms and explore the specific lithium
level indicating the turning point from supporting to impairing effects
on endothelium-dependent vessel relaxation.
5. Conclusion
We conclude that in contrast to the already known endothelial func-
tion stabilizing effect of lithium at low concentrations, higher therapeutic
and supratherapeutic lithium levels impair endothelium-dependent but
not endothelium-independent blood vessel relaxation. Our findings
characterize a differential effect of lithium on the endothelium. These
findings seem to be novel and have two major implications: First, im-
paired endothelium-dependent blood vessel relaxation represents an
additional mechanism contributing to lithium toxicity (e.g. renal and
neurological). And second, effects of lithium on the endothelium are
more complex than previously known and can be completely opposite
even within the narrow therapeutic window of lithium, but also beyond
it. However, future animal and clinical research is needed to further ex-
amine the mechanisms underlying the differential effects of lithium on
endothelium and the vasculature.
Contributors
B. Bosche, T. Noll S. Rej, T. Doeppner and F.V. Härtel, designed the
study. B. Bosche, M. Molcanyi, F.V. Härtel and B. Zatschler performed
the experiments and acquired the data, which B. Bosche, B. Zatschler,
D.J. Müller, S. Rej, J. Hescheler and T. Noll analyzed. B. Bosche, M.
Molcanyi, S. Rej, T Doeppner, D.J. Müller, R.L. Macdonald, T. Noll and
F.V. Härtel wrote the article, which all authors reviewed and approved
for publication.
Conflict of interest
BB got a travel grant and a speaker honorary from CSL Behring,
Germany. RLM is chief scientific officer of Edge Therapeutics, Inc. BB is
a member of the scientific advisory board of Edge Therapeutics. The
other authors declare no conflict of interest.
Acknowledgments
This work was supported by grants of the Deutsche
Forschungsgemeinschaft (DFG) to Dr. B. Bosche (BO 4229/1-1, BO
4229/2-1).
Furthermore, RLM receives grant support from the Physicians Ser-
vices Inc. Foundation, Brain Aneurysm Foundation, Canadian Institutes
for Health Research (CIHR), and Heart and Stroke Foundation of
Canada, SR receives support from a CIHR fellowship award. BB, RLM,
and TN got material support of CSL Behring, Germany and Canada. We
thank Paul J. Turgeon and Matthew S. Yan for their valuable editing on
the manuscript.
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106 B. Bosche et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 67 (2016) 98–106
![cells, too (Ryglewski et al., 2007; Munaron and Fiorio Pla, 2009). There-
by, the endothelium-protective effects of lithium may be important in
cerebrovascular disease, traumatic brain injury with disturbed vascular
regulation (Rajkowska, 2000; Bosche et al., 2003; Dohmen and Bosche
et al., 2007; Gold et al., 2011; Halcomb et al., 2013; Leeds et al., 2014),
and even bipolar disorder (Goldstein and Young, 2013). In endothelial
cells, lithium prevents the discharge of calcium from endogenous stores
by inhibition of the inositol trisphosphate (IP3)-sensitive calcium chan-
nels of the endothelial endoplasmic reticulum (ER) (Schäfer et al.,
2001), thus, counteracting cells stress-induced calcium overload and con-
ferring lithium a cytoprotective potential (Bosche et al., 2013), possibly
through inhibition of GSK-3β (Rej et al., 2015b) and/or IMPase (Chiu
and Chuang, 2010; Dutta et al., 2014). Functionally, maintenance of intra-
cellular calcium homeostasis together with other lithium effects manifest
as modified endothelium-mediated vasodilation (Förstermann and
Münzel, 2006), a potential marker of preserved or improved endothelial
health (Yoo and Kim, 2009; Grove et al., 2015).
In long-term or at supratherapeutic levels, i.e. above the generally
recommended concentrations in humans (Rej et al., 2015a), lithium
can impair particularly kidney and brain function (Laliberté et al.,
2015), where it tends to accumulate (Lichtinger et al., 2013;
Johnson, 1998). Historically, lithium toxicity has been linked to in-
hibition of the GSK-3β or inositol monophosphate pathway leading
to disturbed cellular metabolism (Rybakowski et al., 2013;
Trepiccione and Christensen, 2010) On the other hand, lithium ac-
cumulation or toxicity may also be related to vascular hemodynam-
ic abnormalities (Schou et al., 1968; Laliberté et al., 2015). One
could postulate that the effect of lithium could be harmful to endo-
thelium at high doses and impairs vasodilation, which may contrib-
ute to tissue specific toxicity, e.g. in the brain and kidney (Lichtinger
et al., 2013; Johnson, 1998).
How can lithium be protective to blood vessel endothelium at low
therapeutic doses, but be harmful to blood vessels at higher or
supratherapeutic doses? We hypothesize that at high levels, the effect
of lithium on endothelium reverses — that lithium impairs endothelium-
dependent relaxation of blood vessels. To validate this hypothesis, an
established approach was applied to study vascular function in vessel
grafts (Ebner et al., 2011; Mulvany and Halpern, 1977; Kopaliani et al.,
2014), which recently modified and translated also for human use
(Wilbring et al., 2013).
Yet, to our knowledge, the data presented here uniquely suggested
that supratherapeutic and higher therapeutic, but not lower therapeutic
lithium levels impair particularly endothelium-dependent vascular re-
laxation underlining the concept of opposite effects of lithium at differ-
ent therapeutic concentrations.
2. Material and methods
2.1. Ethics of the animal model
This experimental study was approved by the University Commis-
sion on Animal Experiments with respect to the animal welfare regula-
tions of Germany, in accordance to the European Communities Council
Directive and to the National Institutes of Health (NIH) Guidelines. For
performing the study, written permission was obtained from local
authorities.
2.2. Materials and drugs
All materials, reagents and drugs are described when mentioned
within their respective method sub-sections (see below).
2.3. Cold storage solutions
The used cold storage Tiprotec™ solution (Dr. F. Köhler GmbH,
Bensheim, Germany) contains the following substance concentrations
(all are given in mmol/L): α-ketoglutarate 2, aspartate 5, N-acetyl-histi-
dine 30, glycine 10, alanine 5, tryptophan 2, sucrose 20, glucose 10, Cl−
:
103.1, H2PO4: 1, Na+
: 16, K+
: 93, Mg2+
: 8, Ca2+
: 0.05, deferoxamine:
0.082 and LK 614: 0.017. The pH (at 20 °C) was 7.0 and the osmolarity
was 305 mosmol/L. This standard solution served as the control precon-
ditioning or was supplemented with the different lithium chloride
(LiCl) or lithium acetate (LiAc) concentrations.
2.4. Murine and porcine vessel preparation
The vessel grafts were isolated from murine aortas or from porcine
middle cerebral arteries (MCA). Vessel preparation was performed ac-
cording to the slightly modified method for rodents as described in de-
tail elsewhere (Ebner et al., 2011; Wilbring et al., 2013; Kopaliani et al.,
2014). In brief, male CD57 mice 8 to 10 weeks of age (Charles River Lab-
oratories, Sulzfeld, Germany) were sacrificed by cutting off the upper
cervical spinal cord under deep anesthesia. Male swine (Sus scrofa
domesticus, 24 to 26 weeks of age) were stunned by electroshock and
sacrificed by exsanguination. All mice and swine were dissected imme-
diately. The murine aortas (pars thoracalis without aortic arch) or the
proximal part (M1 segment) of porcine middle cerebral arteries were
recovered and directly placed into a storage solution containing either
1) Tiprotec™ solution only (Dr F. Köhler GmbH, Bensheim, Germany)
or 2) modified Tiprotec™ solution supplemented with 0.4 to
100 mmol/L lithium (LiCl/LiAc, Sigma-Aldrich, Taufkirchen, Germany)
and stored at 4 °C for ≥48 hours (h).
2.5. Post-mortem model of vascular tone control
The post-mortem model with de-nerved vessel grafts was per-
formed to investigate the isolated vessel reaction in response to differ-
ent lithium concentrations independently of the influence of lithium
on the central and thereby also on the vegetative nerve system includ-
ing its remote control of the vessel tone.
2.6. Type of vessel
The thoracic aorta (pars descendens) was chosen as the used vessel
type, because a) it is an elastic type artery containing both the ordinary
vascular smooth muscle cells (SMC) and the myointimal SMC in a rela-
tively high number; moreover, because b) aortic endothelial cells were
used in our previous vessel graft and cell culture studies regarding cyto-
solic [Ca2+
] measurements after long-term and immediate use of lithi-
um and its influence on the specific type of endothelial cells taken
from the aorta (Schäfer et al., 2001; Bosche et al., 2013). Vessel grafts
of the MCA (M1 segment) from porcine brains were additionally used
to investigate the influence of lithium on cerebral endothelium-depen-
dent vasorelaxation.
2.7. Isometric force measurement
After cold storage aortic or cerebral vessel grafts (pipes or long rings,
2 mm in length, 500–600 μm or 1150–1400 μm internal width, respec-
tively) were transferred to a 90%/10% (vol/vol) mixture of phosphate-
buffered saline (PBS) solution and Hank's balanced salt solution
(HBSS, Sigma-Aldrich, Taufkirchen, Germany) gently warmed to
37 °C/98.6 °F over 30 min. Then, the vessel grafts were studied,
stretched with a resting tension equivalent to that obtained by exposure
to an intraluminal pressure of 20 mm Hg for maximal responses. Vessel
rings were equilibrated for 10 min prior to vasomotor analysis. Maximal
contraction was induced by exposure of vessel rings to a potassium-
enriched solution (123.7 mmol/L KCl) and/or to the application of
10 μmol/L phenylephrine (see below Vasoactive Agents). Vessel relaxa-
tion toward acetylcholine (ACH; 10−8.5
to 10−5.5
mol/L) or sodium ni-
troprusside (SNP; 10−8.5
to 10−5.5
mol/L) was tested after a plateau
constriction induced by 10 μmol/L phenylephrine to assess
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