The redox state of transglutaminase 2 controls arterial remodeling.
ABSTRACT While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion, these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall.
Article: Small artery remodeling and erythrocyte deformability in L-NAME-induced hypertension: role of transglutaminases.[show abstract] [hide abstract]
ABSTRACT: Hypertension is associated with inward remodeling of small arteries and decreased erythrocyte deformability, both impairing proper tissue perfusion. We hypothesized that these alterations depend on transglutaminases, cross-linking enzymes present in the vascular wall, monocytes/macrophages and erythrocytes. Wild-type (WT) mice and tissue-type transglutaminase (tTG) knockout (KO) mice received the nitric oxide inhibitor Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME) to induce hypertension. After 1 week, mesenteric arteries from hypertensive WT mice showed a smaller lumen diameter (-6.9 +/- 2.0%, p = 0.024) and a larger wall-to-lumen ratio (11.8 +/- 3.5%, p = 0.012) than controls, whereas inward remodeling was absent in hypertensive tTG KO mice. After 3 weeks, the wall-to-lumen ratio was increased in WT (20.8 +/- 4.8%, p = 0.005) but less so in tTG KO mice (11.7 +/- 4.6%, p = 0.026), and wall stress was normalized in WT but not in tTG KO mice. L-NAME did not influence expression of tTG or an alternative transglutaminase, coagulation factor XIII (FXIII). Suppression of FXIII by macrophage depletion was associated with increased tTG in the presence of L-NAME. L-NAME treatment decreased erythrocyte deformability in the WT mice (-15.3% at 30 dynes/cm(2), p = 0.014) but not in the tTG KO mice. Transglutaminases are involved in small artery inward remodeling and erythrocyte stiffening associated with nitric oxide inhibition-related hypertension.Journal of Vascular Research 02/2008; 45(1):10-8. · 2.65 Impact Factor
Article: Smooth muscle cell changes during flow-related remodeling of rat mesenteric resistance arteries.[show abstract] [hide abstract]
ABSTRACT: To obtain information on the molecular and cellular mechanisms of flow-induced arterial remodeling, we analyzed the morphology and smooth muscle cell (SMC) characteristics in rat mesenteric resistance arteries after interventions that decreased and increased flow. Juvenile male Wistar Kyoto rats were subjected to surgery that, compared with control arteries, provided arteries with chronic low flow and chronic high flow. Low flow resulted in a decreased passive lumen diameter, hypotrophy of the artery wall, and both loss and decreased size of SMCs. Time course studies, with intervention length ranging from 2 to 32 days of altered blood flow, showed that the narrowing of the lumen diameter in low-flow arteries appeared within 2 days and that an early dedifferentiation of SMC phenotype was indicated by markedly reduced levels of desmin mRNA. High flow resulted in an increased passive lumen diameter and in hypertrophy of the artery wall. The hypertrophy resulted from SMC proliferation because SMC number, measured by the 3D-dissector technique, was increased and immunohistochemical assessment of proliferating cell nuclear antigen also showed an increase. The widening of high-flow arteries required 16 days to become established, at which time desmin mRNA was reduced. This time was also required to establish changed wall mass in both low-flow and high-flow arteries. Apoptotic cells detected by TdT-mediated dUTP-biotin nick end labeling staining were mainly located in the medial layer, and evaluation of DNA fragmentation indicated that increased apoptosis occurred in both low flow and high flow. This study shows for the first time direct evidence that reduced and elevated blood flow in resistance arteries produce, respectively, decrease and increase in SMC number, with dedifferentiation of the SMCs in both cases.Circulation Research 08/2001; 89(2):180-6. · 9.49 Impact Factor
Article: Structural properties of rat mesenteric small arteries after 4-wk exposure to elevated or reduced blood flow.[show abstract] [hide abstract]
ABSTRACT: We determined the structure of mesenteric small arteries after chronic elevation and chronic reduction of blood flow. In 6-wk-old rats, we ligated second-order side branches of every other first-order side branch of the superior mesenteric artery. This persistently reduced blood flow (-90%) in the vessels feeding into the ligated trees and elevated blood flow (+80%) in the nonligated mesenteric artery side branches. Four weeks after surgery, vessels that had been exposed to high blood flow (HF) or low blood flow (LF) and vessels from sham-operated rats (Sham) were isolated and mounted in a pressure myograph system. At an intraluminal pressure of 100 mmHg, the internal diameter at rest was larger in HF (533 +/- 23 microm) and smaller in LF (262 +/- 14 microm) than in Sham vessels (427 +/- 15 microm). Also, wall and media cross-sectional areas were larger in HF and smaller in LF than in Sham vessels (media: 22 +/- 1, 11 +/- 2, and 16 +/- 1 x 10(3) microm2, respectively), but circumferential wall stress did not differ among groups. DNA content was significantly increased in HF vessels (+100%) and was not modified in LF vessels. Maximal vasoconstrictions elicited by high potassium or norepinephrine were slightly increased in HF vessels but were reduced by 50% in LF vessels. Thus chronic changes in blood flow give rise to structural changes that normalize circumferential wall stress. Elevated blood flow resulted in outward hypertrophic remodeling involving hyperplasia. Reduced blood flow resulted in inward hypotrophic remodeling accompanied by hyporeactivity of the arterial smooth muscle.The American journal of physiology 11/1997; 273(4 Pt 2):H1699-706.
The Redox State of Transglutaminase 2 Controls Arterial
Jeroen van den Akker1, Ed VanBavel1, Remon van Geel2, Hanke L. Matlung1, Bilge Guvenc Tuna1,
George M. C. Janssen3,4, Peter A. van Veelen3, Wilbert C. Boelens2, Jo G. R. De Mey5, Erik N. T. P. Bakker1*
1Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 2Department of Biomolecular
Chemistry 271, Nijmegen Center for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands, 3Department of Immunohematology and Blood
Transfusion, Leiden University Medical Centre, Leiden, The Netherlands, 4Netherlands Proteomics Centre, Utrecht, The Netherlands, 5Department of Pharmacology and
Toxicology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction
depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation
of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward
remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the
presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of
mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced
translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward
remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the
nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking
substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with
the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion,
these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results
from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall.
Citation: van den Akker J, VanBavel E, van Geel R, Matlung HL, Guvenc Tuna B, et al. (2011) The Redox State of Transglutaminase 2 Controls Arterial
Remodeling. PLoS ONE 6(8): e23067. doi:10.1371/journal.pone.0023067
Editor: Qingbo Xu, King’s College London, University of London, United Kingdom
Received May 4, 2011; Accepted July 6, 2011; Published August 25, 2011
Copyright: ? 2011 van den Akker et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by the Netherlands Heart Foundation (grant NHS.2005.B080 to Jeroen van den Akker and NHS.2001.T038 to Hanke L. Matlung).
Ed VanBavel and Jo G.R. De Mey are supported by the Dutch Top Institute Pharma, TIPharma T2-108. Bilge Guvenc Tuna is supported by the European Union,
Marie Curie ITN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Ed VanBavel and Jo G.R. De Mey are supported by the Dutch Top Institute Pharma. This is a consortium of Abbott, Academic Medical
Center (AMC) Amsterdam, Leiden University Medical Center, Maastricht University, VU University Medical Center. This does not alter the authors’ adherence to all
the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
Small arteries represent the main site of resistance in the
vascular system, and as such, have a large impact on tissue
perfusion and blood pressure. Inward remodeling of small arteries
occurs after a reduction in blood flow, but is also associated with
high blood pressure [1–3]. It is a hallmark of essential
hypertension and a strong predictor of cardiovascular events .
Previous work from our group showed that transglutaminases, in
particular type 2 transglutaminase (TG2), play a crucial role in the
inward remodeling of small arteries after reduced blood flow and
hypertension in vivo, and chronic vasoconstriction in vitro [5–11].
The mechanism by which TG2 contributes to vascular remodeling
however, remains poorly understood.
One reason for the elusive role of TG2 in remodeling may relate
to its wide range of actions. Its best known function is the
stabilization of matrix proteins through the formation of a specific
cross-link, the Ne(c-glutamyl)lysine isopeptide bond, which is the
result of a transamidation reaction . This reaction is regulated
by several factors including calcium, GTP, nitric oxide and the
redox potential [13,14]. The isopeptide bond provides mechanical
strength and resistance to proteolytic degradation in tissues.
Recent work from Santhanam et al  showed that the cross-
linking action of TG2 is highly relevant for human cardiovascular
pathology. Thus, these authors showed that the formation of cross-
links by TG2 relates to the stiffening of large arteries that is
associated with aging. Besides the formation of isopeptide bonds
within and between proteins , TG2 promotes cell adhesion via
its binding to fibronectin, integrin a5b1 and heparan sulfate
proteoglycans [16,17]. In addition, TG2 acts as protein disulfide
isomerase , functions as a G-protein  and aids in the
regulation of cytoskeletal structure and cell contractility [20–22].
Of great potential relevance for cardiovascular pathology,
Stamnaes et al.  recently showed that TG2 contains a triad
of cysteine residues that act as a redox sensor, which could regulate
the cross-linking activity of TG2 in the extracellular environment.
In the present study we investigated the activation, (sub) cellular
localization, and substrates of TG2 during the inward remodeling
of small arteries. Besides the regulation of TG2 by calcium and
nitric oxide, we focused on the redox state of TG2 using reducing
agents with different properties regarding cellular permeability.
We report that TG2 is translocated to the surface of smooth
PLoS ONE | www.plosone.org1 August 2011 | Volume 6 | Issue 8 | e23067
muscle cells (SMCs) upon intracellular reduction. In a reduced
state TG2 acts as a membrane-bound cross-linking enzyme in
SMC, which coincides with inward remodeling. Using mass
spectrometry, we identified several matrix proteins as TG2
substrates. Taken together, these data elucidate the regulation,
secretion and substrates of TG2 in the process of vascular
Mice and vessel isolation
Four months old male C57Bl/6 mice (Harlan) were anesthetized
using isoflurane and sacrificed by cervical dislocation. Then, the
abdomen was opened and the mesentery was excised and placed in
cold MOPS buffer. First and second order arteries were isolated
from the mesenteric vasculature. Arteries were cut in equal-sized
pieces where one segment was randomly assigned as control and the
other segments subjected to various interventions. This approach
allowed for pair-wise statistics and drastically decreased variability
which results from anatomical variation in vessel caliber. All
protocols consisted of a 24-hour incubation period at 37uC, where
the vessels were placed in 100 mL buffer containing Leibovitz
medium with 10% fetal bovine serum (Gibco), a mix of antibiotic-
on the specific protocol. All experiments were approved by the
Committee for Animal Experiments of the Academic Medical
Center Amsterdam (permissions 101221 and 101555). TG2
knockout mice were obtained from Prof. G. Melino (Rome, Italy)
and bred at our local facility.
Pressure myograph and remodeling
To determine remodeling of the arteries, segments were
cannulated in a pressure myograph system after the incubation
period and inner diameters were recorded as described previously
. After checking for leaks, a passive pressure- diameter
relationship was determined in calcium-free MOPS buffer,
supplemented with papaverine (0.1 mmol/L) to rule out influences
of vasomotor tone.
Exogenous recombinant TG2
In this set of experiments, vessels were exposed to recombinant
human TG2 (Zedira, T002) or cross-linking deficient C277S-TG2
(Zedira, T018), with or without the membrane-impermeable
(TCEP, 1 mmol/L). Control experiments were included were
segments only were exposed to TCEP. In all experiments,
recombinant TG2 was administered at 50 mg/mL.
Activation of endogenous TG2
In this set of experiments, vessels were incubated with calcium
ionophore A23187 (Sigma, C7522: 1 mmol/L). Alternatively,
vessels received 2 mmol/L DTT (Sigma, 43816), which is
membrane-permeable. In the latter experiment, TG2 activity
was blocked using either the TG2 active-site inhibitor L682777
(Zedira, T101: 10 mmol/L, also known as R283) or the NO donor
SNAP (Sigma, N3398: 1 mmol/L).
The effect of DTT on vessel viability was assessed in a separate
set of vessels. Here, the contractile response to the thromboxane/
prostaglandin agonist U46619 (Sigma, D8174: 1 mmol/L) was
measured after a 24-hour incubation with 2 mmol/L DTT.
Localization of TG2 activity
In vessels stimulated with calcium ionophore or DTT, TG2
activity was visualized using the pseudo-substrate cadaverine,
linked to either FITC (AnaSpec, 81504; 100 mmol/L) or
AlexaFluor594 (Invitrogen, A-30678;10 mmol/L). In experiments
where SNAP was used to inhibit TG2 activity, cadaverine was
added .30 min after SNAP. SNAP was used at 1024mol/L with
calcium ionophore and at a concentration of 1023mol/L with
DTT. Vessels were fixed with formalin, mounted on glass slides
using Vectashield/DAPI (Vector Laboratories H-1500) and
imaged on a confocal microscope (Leica TCS SP2). TG2 activity
was quantified by spatial integration of FITC or AlexaFluor594
signal in ImageJ. Data were corrected for vessel size and depicted
in arbitrary units.
Immunostaining of TG2
The effect of DTT on the translocation of TG2 was assessed by
immunofluorescent staining of extracellular TG2 on cultured
mouse smooth muscle cells (MOVAS, ATCC CRL-2797). Cells
were grown in microscopic culture chambers (BD Falcon 354102,
untreated glass) that were coated with fibronectin. After a culture
period of 24 hours in DMEM with 10% FCS, 0.1 mmol/L DTT
was added for 2 hrs. Then cells were washed 3 times with warm
PBS and fixated with cold formalin. After blocking with BSA/goat
serum, the non-permeabilized cells were stained with a rabbit
polyclonal TG2 antibody Ab-4 (Neomarkers RB-060-P, 1:10; 1 hr
at room temperature) followed by anti-rabbit Cy3 (Brunschwig
111-165-144, 1:200; 1 hr at room temperature) as secondary
antibody, and slides were mounted in Vectashield/DAPI (Vector
Substrates of transglutaminase in smooth muscle cells
The substrates for transamidation catalyzed by TG2 were
determined using a mouse smooth muscle cell line (MOVAS,
ATCC CRL-2797). Cells were cultured in DMEM with 10% FCS
for 96 hours in T75 flasks. Then either BPA (biotinylated
pentylamine, Invitrogen A1594, 1 mmol/L) or Q-peptide (Bio-
tin-GQEPVR, synthesized using standard Fmoc-based solid phase
peptide synthesis, 0.25 mmol/L) were added to function as lysine
and glutamine donors respectively , while a control group was
left without competitive substrate. All groups received 0.1 mmol/L
DTT to increase the amount of active TG2 in the extracellular
matrix. After a 24 hrs incubation period, the cultures were washed
3 times with warm PBS to remove non-bound BPA and Q-
peptide. Then the lysates were collected in a 1% SDS solution and
boiled for 5 minutes at 95uC to denature the proteins. For each
group, the material from 2 T75 flasks was pooled and stored at
220uC until further use.
Lysates were sonicated (5 times 30 seconds at room tempera-
ture) and dialysed against 1% SDS (3 times 500 mL for 1 hour at
room temperature) to further decrease the amount of non-bound
BPA and Q-peptide. Then lysates were centrifuged at 15.700 g for
10 minutes at room temperature and 13 mL buffer containing
100 mmol/L NaCl, 50 mmol/L Tris HCl pH 7.5, 1 mmol/L
EDTA, 0.5% NP40 was added to 1.2 mL supernatant. Strepta-
vidin-Agarose (50 mL, Sigma S1638) was added and the mixture
was rotated end-over-end at room temperature for 20 hours. The
agarose-beads were washed with 100 mmol/L NaCl, 50 mmol/L
Tris HCl pH 7.5, 1 mmol/L EDTA, 0.05% NP40 (3 times
5 minutes end-over-end at room temperature) and taken up in 2x
sample buffer (4% SDS, 10% b-mercaptoethanol, 20% glycerol,
0.06% bromophenol blue and 0.5 mol/L Tris HCl pH 6.8).
For Western blot analysis 2% of each pull-down was loaded on
a 12% SDS-PAGE gel and after blotting stained with IRdye
800CW Streptavidin (li-Cor), rabbit polyclonal anti-Fibronectin
(GIBCO 1A0540) and rabbit polyclonal nidogen-1 (Immun
Transglutaminase in Arterial Remodeling
PLoS ONE | www.plosone.org2August 2011 | Volume 6 | Issue 8 | e23067
For MS analysis 20% of each pull-down was loaded on a 12%
SDS-PAGE gel and stained with colloidal Coomassie Brilliant
Blue. The biotin containing region (,30 kDa and up) from each
Coomassie stained lane was sliced into 16 equal parts. Proteins in
the slice were reduced with DTT, alkylated with iodoacetamide
and digested with trypsin using the Proteineer DP digestion robot
(Bruker, Bremen, Germany), adapted in house to accommodate
larger gel pieces. The tryptic peptides were extracted from the gel,
lyophilized, dissolved in 95/3/0.1 v/v/v water/acetonitril/formic
acid and subsequently analyzed by on-line nanoHPLC MS/MS
using a1100 HPLC system (Agilent Technologies), as previously
described . Peptides were trapped at 10 mL/min on a 15-mm
column (100-mm ID; ReproSil-Pur C18-AQ, 3 mm, Dr. Maisch
GmbH) and eluted to a 200 mm column (50-mm ID; ReproSil-Pur
C18-AQ, 3 mm) at 150 nL/min. All columns were packed in
house. The column was developed with a 120-min gradient from 0
to 30% acetonitrile in 0.1% formic acid. The end of the
nanocolumn was drawn to a tip (ID ,5 mm), from which the
eluent was sprayed into a 7-tesla LTQ-FT Ultra mass spectrom-
eter (Thermo Electron). The mass spectrometer was operated in
data-dependent mode, automatically switching between MS and
MS/MS acquisition. Full scan MS spectra were acquired in the
FT-ICR with a resolution of 25,000 at a target value of 3,000,000.
The two most intense ions were then isolated for accurate mass
measurements by a selected ion monitoring scan in FT-ICR with a
resolution of 50,000 at a target accumulation value of 50,000.
Selected ions were fragmented in the linear ion trap using
collision-induced dissociation at a target value of 10,000. In a post-
analysis process, raw data were first converted to peak lists using
Bioworks Browser software v 3.2 (Thermo Electron), then
submitted to the SwissProt database version 51.6 using Mascot
v. 2.2.04 (www.matrixscience.com) for protein identification and
finally sorted and compared using Scaffold software version 3.0.1
(www.proteomesoftware.com). Mascot searches were with 2 ppm
and 0.8 Da deviation for precursor and fragment mass, respec-
tively, and trypsin as enzyme. Scaffold filtered for identified
proteins with at least 2 peptides with 95% confidence. Collision-
induced dissociation spectra were also manually inspected.
Common contaminants were removed manually from the list.
Regulators of redox balance during inward remodeling
The expression of redox regulating enzymes in inwardly
remodeling vessels was investigated using a microarray approach
published previously by Wesselman et al.  In short, flow-
modifying surgery was performed on rat first-order mesenteric
arteries. This leads to a flow reduction in ligated vessels to
approximately 10% of control, and a doubling of flow in adjacent
high flow vessels. Animals were sacrificed after 1, 2, or 4 days and
for each time point vessels from 4 animals were pooled. cDNA
from either low or high flow vessels linked to a Cy5 probe was
hybridized onto 2 different microarrays in the presence of Cy3-
labeled cDNA from control vessels. Up- or down regulation of a
number of redox regulating enzymes with decreased flow was then
normalized to control values. If genes were present more than
once in the 2 arrays employed, their values were averaged.
Data are shown as mean 6 SEM. For all measurements of P,d-
curves, differences in diameter between groups were tested at each
pressure level using a paired T-test with Bonferroni correction
when appropriate. Distensibility was calculated as the lumen
diameter of the artery at 120 mmHg divided by the diameter at
5 mmHg. For quantification of fluorescence, 3 images were
averaged per vessel. In all figures, P-values smaller than 0.05 resp.
0.01 are indicated by single or double symbols (e.g. * and **).
Vascular remodeling by recombinant TG2 requires a
Small mesenteric arteries were incubated for 24 hours under
of these vessels was determined by cannulation and recording of a
passive pressure-diameter (P,d) relationship. Incubation of mesen-
teric arteries with exogenous recombinant TG2 had no effect on the
P,d-curve as compared to untreated control arteries (Figure 1A).
Distensibility of the arteries, determined as the ratio of the diameter
at 120 mmHg divided by the diameter at 5 mmHg, was
unchanged: 1.9860.14 vs. 1.9360.10 for control and TG2
respectively. Administration of 1 mmol/L TCEP, a cell-imperme-
able reducing agent, also did not affect the P,d curve (Figure 1B).
Distensibility was unchanged: 2.0160.12 vs. 2.0360.11 for control
and TCEP respectively. However, when TG2 was added together
with TCEP, vessel diameter was significantly reduced at higher
pressure levels (Figure 1C). This resulted in a significant decrease in
distensibility from 1.9560.04 to 1.7660.14 for TCEP vs.
TCEP+TG2 (p=0.01). When the catalytically inactive TG2
mutant Cys-277 was used instead of recombinant TG2, inward
remodeling was again absent (Figure 1C). Distensibility was similar
to TCEP alone: 1.9260.08 (NS). These data therefore show that
inward remodeling by TG2, characterized by a reduction in
distensibility, depends on its cross-linking action and requires the
presence of a reducing agent.
A cell-permeable reducing agent activates endogenous
In order to test if endogenous TG2 could be activated by a cell-
permeable reducing agent, vessels were incubated with dithiothre-
itol (DTT). As indicated by FITC-cadaverine incorporation, DTT
induced a profound increase in TG2 activity in the vessel wall
(Figure 2A). This activity was completely abolished in vessels
incubated with the TG2 inhibitor L682777 (Figure 2, panels A–B).
The activation of endogenous TG2 with DTT caused a highly
significant inward remodeling, which was almost completely
prevented by L682777 (Figure 2C). The effect of endogenous
TG2 on vascular remodeling was similar to that of exogenous
TG2, with a reduction in vessel diameter at higher distending
pressures only (Figure 2C). Thus, distensibility decreased from
2.0060.12 to 1.7160.08 for control and DTT respectively
When small mesenteric arteries from TG2 knockout mice were
exposed to DTT, inward remodeling was observed also, albeit to a
lesser extent as compared to vessels from C57BL/6 mice.
1.9560.08 for control and DTT respectively (p,0.001). In this
case however, the TG2 inhibitor L682777 was ineffective.
Distensibility was not altered as compared to DTT: 1.9760.11
(NS). These data suggest that in the TG2 knockout mice other
mechanisms are activated by DTT which induce remodeling
In subsequent experiments we tested if remodeling induced by
DTT could also be counteracted by nitric oxide. Transglutamin-
ase activity was strongly reduced by the NO donor SNAP, as
indicated by the incorporation of Alexa Fluor-594/cadaverine
(Figure 3, panels A–B). This was paralleled by an almost complete
inhibition of the remodeling (Figure 3C). Distensibility decreased
significantly by treatment with DTT from 1.9660.10 to
Transglutaminase in Arterial Remodeling
PLoS ONE | www.plosone.org3 August 2011 | Volume 6 | Issue 8 | e23067
1.6460.13 (p,0.01), but was reversed by SNAP to 1.8660.06
(p=0.01). Control experiments showed that SNAP alone did not
affect vessel properties (Figure S2). Hence, these results show that a
shift of the intracellular redox balance to a more reduced state
activates a TG2-dependent inward remodeling and, in addition,
this remodeling can be inhibited by nitric oxide (NO).
Calcium ionophore A23187 stimulates TG2 activity but
does not induce inward remodeling
Calcium triggers a conformational change in TG2, providing
access of substrates to TG2’s active site . Normally, under
Figure 1. Microvascular remodeling by transglutaminase
requires a reducing agent and cross-linking activity. Passive
pressure-diameter relationships (P,d curve) of isolated, cannulated
arteries were measured to reveal remodeling. (A) Exogenous TG2 had
no effect on vessel properties in the absence of a reducing agent. (B)
The cell-impermeable reducing agent TCEP did not induce remodeling
by itself. (C) When exogenous TG2 was combined with TCEP, inward
remodeling was observed. This effect was absent with recombinant
TG2, defective in cross-linking. Data were averaged over 6 vessels
obtained from 3 mice. ** TCEP vs. TG2+TCEP: P,0.01.
Figure 2. Activation of endogenous TG2 by the cell-permeable
reducing agent DTT. (A–B) Exposure to DTT induces TG2 activity in
the medial layer of the vessel, as shown by the incorporation of FITC
cadaverine. (C) TG2 activation with DTT induces inward remodeling, as
indicated by a downward shift of the P,d-curve. This was blocked with a
site-specific TG2 inhibitor (L682777). Data were averaged over 6 vessels
obtained from 3 mice, with 3 images per vessel; scalebar=75 mm.
* non-stimulated vs. DTT: P,0.05, ** P,0.01, # DTT vs. L682777+DTT:
P,0.05, ## P,0.01.
Transglutaminase in Arterial Remodeling
PLoS ONE | www.plosone.org4August 2011 | Volume 6 | Issue 8 | e23067
physiological conditions the level of intracellular calcium is too low
to induce cross-linking activity by TG2. However, after exposure
to vasoconstrictor substances, or under pathological conditions,
the level of intracellular calcium may rise considerably. To test the
role of calcium we analyzed the effect of the calcium ionophore
A23187 on TG2 activity and arterial remodeling. Administration
of the ionophore increased TG2 activity in the vessel wall about 3-
fold, as demonstrated by the incorporation of FITC-cadaverine
(Figure 4, panels A,B). This calcium-induced intracellular activity
could be inhibited by 1 mmol/L of the NO donor SNAP (Fig. 4,
panels A,B). Despite the increase in TG2 activity, the calcium
ionophore did not induce small artery inward remodeling, as
reflected by an unchanged P-d curve after incubation of
cannulated arteries with 1 mmol/L A23187 (Figure 4C). Disten-
sibility was also unchanged: 1.9060.14 vs. 1.9460.13 for control
and A23187 respectively (NS).
Localization of TG2 activity is stimulus-dependent
While activation of intracellular TG2 with DTT caused
significant inward remodeling, this was absent for activation by
the calcium ionophore A23187. We tested whether this difference is
related to localization of TG2 activity. In the adventitia, TG2
activity was virtually absent (Video S1). The incorporationof FITC-
cadaverine around endothelial cells was also relatively low. In
contrast, TG2 activity following stimulation with A23187 or DTT
was prominent in the medial layer. The subcellular staining pattern
in the SMCs was strongly dependent on the stimulus for TG2
activation. Following stimulation with the calcium ionophore, TG2
activity appeared throughout the cytosol, while cellular boundaries
remained clearly visible (Figure 5). On the other hand, DTT
induced TG2 activity at the cell membrane, producing a mirror
activity at the membrane, patches of high TG2 activity were
observed at the interface of the endothelium and smooth muscle
layers (Video S1). Thus, a clear difference was observed in the
localization of TG2 activity with A23187 and DTT.
Intracellular reduction increases TG2 protein at the cell
The confocal images showed a membrane-bound TG2 activity
upon stimulation with DTT, but did not provide sufficient
resolution to determine whether the activity is intra- or
extracellular. We therefore studied the presence of TG2 on non-
permeabilized smooth muscle cells with immunocytochemistry.
Cultured SMCs were stimulated with a low dose of DTT and
stained for extracellular TG2. This revealed a strong increase in
the level of extracellular TG2 as compared to untreated control
cells (Figure 6). Hence, these data suggest that a reduced state
triggers translocation of TG2 from the cytosol to the cell surface.
Substrates of TG2
As TG2 activity at the smooth muscle cell membrane was
associated with inward remodeling, we next investigated the
extracellular substrates of TG2 using cultured SMCs that were
stimulated with DTT. Labeling of TG2 substrates with the lysine
donor biotinylated pentylamine (BPA) or a specifically designed Q-
peptide (glutamine donor) was followed by a pull down assay and
mass spectrometry. This revealed a number of proteins as substrate
for TG2 (Table 1). Fibronectin was the major extracellular
substrate, cross-linked both to BPA and Q-peptide. In addition,
BPA identified the ECM components collagen, fibulin-2 and
nidogen-1 as glutamine donors for transamidation. Both fibronectin
and nidogen-1 were subsequently confirmed as TG2 substrates in
western blots (Figure S3). Furthermore, a number of cytoskeletal
and other intracellular proteins were identified.
Regulators of redox balance during in vivo remodeling
The activation of TG2 by DTT proved to be a strong stimulus
for inward remodeling, but represents an artificial means of
Figure 3. DTT-induced activation of TG2 can be counteracted
with the NO donor SNAP. (A–B) Incorporation of AlexaFluor594/
cadaverine was significantly inhibited with SNAP. (C) SNAP abolished
the inward remodeling induced by DTT. Data were averaged over 6
vessels obtained from 3 mice, with 3 images per vessel. Scale-
bar=75 mm. * non-stimulated vs. DTT: P,0.05, ** P,0.01, # DTT vs.
SNAP+DTT: P,0.05, ## P,0.01.
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manipulating the redox state. Therefore, the expression of several
enzymes capable of reducing thiol groups on proteins was studied
in vessels remodeling in vivo. The mRNA expression was assessed
in vessels stimulated to remodel inwardly by reducing blood flow
in vivo. In arteries undergoing inward remodeling, selenoprotein P
upregulation peaked after 2 days (Table 2). Glutathione transferase
was already increased 80% after 24 hrs, followed by a gradual
decline of expression. In outward remodeling vessels on the other
hand, these enzymes were downregulated after 1 and 2 days (data
Vascular remodeling of small arteries after reduced blood flow,
hypertension, and exposure to vasoconstrictors depends on TG2
[7,8,11]. In large arteries TG2 is involved in vascular calcification,
and atherosclerotic plaque development and stability [27–31].
Recently, also large artery stiffening associated with aging was
shown to be dependent on TG2 . While many studies have
contributed to the understanding of the regulation and functions of
TG2 , the actual role of TG2 in vascular remodeling remained
largely unknown. In our previous work we identified a strong
relationship between vascular tone and remodeling. We reported
that persistent vasoconstriction induces inward remodeling in
several types of arteries [11,32,33]. This remodeling could be
inhibited or reversed by vasodilator compounds such as the
calcium channel inhibitors verapamil  or amlodipine . As
constriction and dilation mechanisms act partly through modula-
tion of intracellular calcium levels, we herein tested the hypothesis
that elevation of intracellular calcium triggers TG2 activity and
remodeling. The calcium ionophore indeed increased intracellular
transglutaminase cross-linking activity, but remodeling was
completely absent. Thus, although intracellular proteins might
have been cross-linked, this did not affect vessel caliber (Figure 4).
Thus, these results suggested that other mechanisms of TG2
activation than elevation of intracellular calcium operate during
We found that the redox state of TG2 is a critical determinant
in small artery remodeling. DTT induced a strong inward
remodeling response, which was inhibited by the TG2 inhibitor
(Figure 2). The data on remodeling by exogenous recombinant
TG2 (Figure 1) underline the need for a reduced environment. As
the cell-impermeable reducing agent TCEP did not change blood
vessel diameter, the remodeling most likely stems from activation
of an intracellular source of TG2. Since transglutaminase activity
associated with remodeling was located at the surface of smooth
muscle cells, we inferred that upon intracellular reduction, TG2 is
excreted but remains bound to the cell membrane. This was
substantiated by specific staining of extracellular TG2, using
cultured SMCs. Similar mechanisms may exist in endothelial cells
. We speculate that after translocation to the cell membrane,
TG2 activity is fully uncovered by the high extracellular calcium
The reversible formation of disulfide bridges, S-nitrosylation
and S-glutathiolation of key cysteines are increasingly recognized
as crucial mediators of protein activation and function [34–36].
TG2 is a good example of such regulation. It contains 18
sulfhydryl residues which can potentially be oxidized to form
disulfide bridges, thereby impairing TG2 function [23,37,38]. The
active site cysteine (Cys-277) was shown not to be easily prone to
form disulfide bridges, probably due to the low accessibility of the
active cysteine [23,37,39,40]. However, disulfide bonds formed
using cysteine residues from other parts of TG2 have been shown
to strongly reduce cross-linking activity as well [37,40,41].
Recently, a crucial cysteine pair at amino acids 370–371,
controlled by Cys-230, was identified . It has been
hypothesized that these disulfide bridges impede the calcium-
triggered conformational change that is required for activation
[23,37]. Thus, a reduced state is necessary for TG2 to allow its
cross-linking action. Generally speaking, the intracellular com-
Figure 4. Incubation with calcium ionophore (A23187) induces
transglutaminase activity without remodeling. (A–B) Exposure to
A23187 stimulates the incorporation of FITC cadaverine. TG2 activity
can be inhibited by the NO donor SNAP. (C) A23187 does not induce
inward remodeling. Data were averaged over 6 vessels obtained from 3
mice, with 3 images per vessel. Scalebar=75 mm. * non-stimulated vs.
A23187: P,0.05, # A23187 vs. A23187+SNAP: P,0.05.
Transglutaminase in Arterial Remodeling
PLoS ONE | www.plosone.org6August 2011 | Volume 6 | Issue 8 | e23067
partment is relatively reduced, whereas the extracellular environ-
ment is more oxidized . More detailed studies have revealed
that the redox balance is further controlled at the subcellular level
[42–44]. In addition, the redox state of proteins can be
individually regulated . The cell-permeable reductant DTT
that we used is known to activate in situ transamidation without
affecting other TG2 functions . We recognize that DTT is a
non-specific reducing agent that might exert toxic effects.
However, incubation with DTT did not affect vessel viability,
assessed by its ability to contract (Figure S4). Hence, DTT did not
have an overt detrimental effect on the used ex vivo vessels. In
addition, the concentration of DTT that was used appears
relatively high, but one needs to keep in mind that intracellular
reducing compounds such as glutathione are present in the
millimolar range .
The observation that a reduced state of TG2 is essential for its
cross-linking activity, seems to contradict a report that TG2 is
activated by oxidative stress via reactive oxygen species (ROS)
. However, UV irradiation or administration of exogenous
H2O2did not potentiate in vitro TG activity in a large number of
cell types [14,48]. Therefore, TG2 activity in response to ROS
may be a secondary effect, possibly linked to calcium leakage over
the damaged cell membrane or increased TG2 expression in
apoptosis-prone cells [14,49,50]. Indeed, cytosolic ROS were
reported to trigger an increase in cytosolic calcium . In
addition, generation of ROS was reported to inhibit TG2
In addition to disulfide formation, TG2 cross-linking activity
can be regulated by nitrosylation of cysteines . Here, the redox
balance plays an important role as well: cationic nitric oxide causes
S-nitrosylation, but anionic NO leads to formation of a disulfide
bridge . In the present study, we showed that activation of
TG2 within the vessel wall, either by reduction or elevated
intracellular calcium is inhibited by NO. The inhibition of TG2
activity fully prevented inward remodeling. As the nitric oxide
level falls in several physiological and pathological conditions, such
as low blood flow, hypertension and aging, this may be an
important determinant in transglutaminase activity in vivo.
Indeed, we previously showed that inhibition of NO synthesis
results in TG2 dependent inward remodeling [1,53]. In further
support, recent work by Santhanam et al.  showed that
vascular stiffening associated with aging depends on TG2 activity
and a reduction in nitric oxide levels.
The substrates of TG2 in small artery remodeling have not been
previously studied. We identified fibronectin as both a lysine and
Figure 5. Differential localization of transglutaminase activity. (A) When vessels were incubated with the cell-permeable reducing agent DTT,
FITC cadaverine was cross-linked at the cell membrane of smooth muscle cells. (B) When vessels were incubated with the calcium ionophore A23187,
FITC cadaverine appeared throughout the cytosol. Vascular remodeling occurred with DTT treatment, but not with the calcium ionophore.
Figure 6. Intracellular reduction increases cell surface TG2.
Incubation of cultured smooth muscle cells with DTT (0,1 mmol/L)
increased the amount of extracellular TG2 (shown in red), as visualized
by immunostaining of non-permeabilized cells. Fluorescence intensity
was integrated per cell and averaged over 35–37 cells per group.
Scalebar=20 mm. ** non-stimulated vs. DTT: P,0.01.
Transglutaminase in Arterial Remodeling
PLoS ONE | www.plosone.org7August 2011 | Volume 6 | Issue 8 | e23067
glutamine donor (Table 1). Another relevant extracellular
substrate is fibulin-2, which is active in tissue remodeling by
cross-linking several elements in the pericellular matrix . The
confocal images showed high TG2 activity at the interface
between smooth muscle cells and endothelial cells (Video S1).
This layer, the internal elastic lamina, contains laminin-nidogen
complexes and collagen type IV, which are both known substrates
for TG2 [55,56]. We confirmed nidogen-1 as substrate for SMC-
derived TG2 using BPA. Regarding the collagen family, the alpha-
1 chain of collagen type I was detected as TG2 substrate. Thus,
although we identified a number of proteins that are cross-linked
and known to determine extracellular matrix properties, the actual
substrate(s) responsible for remodeling remains to be determined.
Also, the relevance of the intracellular substrates, including
cytoskeletal proteins and glycolytic enzymes, warrants further
An important question is how reduction of specific proteins such
as TG2 is achieved in vivo. We addressed this question by studying
mRNA expression of redox-related proteins using a micro-array
approach. Here we found several reducing enzymes to be quickly
upregulated in vessels undergoing inward remodeling (Table 2).
Glutathione transferase (GST), which catalyzes the conjugation of
reduced glutathione to substrate proteins, was strongly upregu-
lated after 1 and 2 days, together with glutathione reductase.
Interestingly, a broad protein interaction study identified GST as
the 2ndmost important interaction partner of immobilized TG2
, a finding confirmed in several pathological conditions [57–
59]. Thioredoxin is expressed amongst others in endothelial and
SMCs, was detected in plasma [60,61] and is involved in protein
denitrosylation . Surprisingly, thioredoxin was downregulated
in inward remodeling vessels, although its corresponding reductase
was slightly upregulated at all time points. On the other hand, the
expression of selenoprotein P, which is structurally closely related
to thioredoxin reductase, was elevated 174% after 2 days.
Although protein disulfide isomerase was reported to act as
reductase at the cell membrane of SMCs , its expression level
was unchanged. Macrophage migration inhibitory factor, which
plays a role during inflammation but also in redox regulation at
the cell membrane , was upregulated at all time points. Taken
together, the upregulation of a panel of reducing enzymes was
identified in vessels in the process of inward remodeling. These
enzymes could provide a more reduced state necessary for TG2
In summary, using the Cys-277 mutant of TG2 and an active-
site inhibitor, we showed that the cross-linking action of TG2 is
necessary to induce inward remodeling of small arteries.
Importantly, TG2 needs to be kept in a reduced state to fulfill
Table 1. TG2 substrates in cultured smooth muscle cells.
control BPAQ-peptide known
fibronectin precursor 6347 yes
collagen alpha-1 chain precursor41yes
Cytoskeleton & Cell Membrane
vimentin614 13 yes
actin3 1412 yes
tubulin alpha-1175 no
tubulin beta-5 73 yes
WD repeat protein 12 no
annexin A12 no
L-lactate dehydrogenase2 106no
DNA, RNA & Protein Synthesis
elongation factor 174yes
histone H1.2 5yes
initiation factor 4A-I133 no
ribosomal protein 53-A23 no
elongation factor 213no
heat shock protein 9074 yes
guanine nucleotide-binding protein53 no
serum albumin precursor14 no
multifunctional protein ADE22 no
Cultured smooth muscle cells were incubated with BPA and Q-peptide, which
were used as lysine and glutamine donor respectively. Samples were purified by
a streptavidin pull down assay and bands were analyzed by mass spectrometry.
Values represent the number of unique peptides found for each protein and
give an indication of the abundance of the protein in the sample.
1Database used: http://genomics.dote.hu/wiki/index.php/
Table 2. Changes in mRNA of redox regulating enzymes
during inward remodeling.
Glutathione Reductase4030 101
Glutathione Transferase T1 8050
Macrophage Migration Inhibitory Factor57 13703
Protein Disulphide Isomerase0
Selenoprotein P 28174 625
Thioredoxin Reductase20 2020 1
Xanthine Dehydrogenase73 90473
Inward remodeling was induced by a surgically imposed decrease in blood flow
in rat mesenteric arteries. Vessels were harvested at several time points and
mRNA expression was determined by microarray analysis. Data represent
changes in mRNA expression as percentage from control. An increased
expression of several redox regulating enzymes was found. For each time point,
vessels from 4 animals were pooled. Some genes were present several times on
Transglutaminase in Arterial Remodeling
PLoS ONE | www.plosone.org 8August 2011 | Volume 6 | Issue 8 | e23067
its action. Within the vessel wall, we found that smooth muscle
cells respond to intracellular reduction with a strong increase in
TG2 activity at the cell membrane. This localized transglutamin-
ase activity was associated with inward remodeling. Of physiolog-
ical and pathological relevance, TG2 activity and remodeling
could be inhibited by addition of a NO-donor. A schematic
overview of the results is given in Figure S5. Using mass
spectrometry, we identified a number of proteins as substrates
for TG2 that could be responsible for the change in vessel caliber.
Finally, we found several reducing enzymes that were strongly
upregulated in vessels undergoing inward remodeling induced by
low blood flow. These enzymes could help to provide a more
reduced state during inward remodeling in vivo.
mice. Passive pressure-diameter relationships of isolated, cannu-
lated arteries from TG2 knockout mice that were exposed to DTT
showed inward remodeling. In contrast to arteries from C57BL/6
mice, the remodeling of arteries from TG2 knockout mice was not
sensitive to the TG2 inhibitor L682777. *** Indicates P,0.001 for
control vs. DTT.
Effect of DTT on arteries from TG2 knockout
pressure-diameter relationships of isolated, cannulated arteries
were not altered after 24 h exposure to the nitric oxide donor
SNAP does not alter vessel properties. Passive
TG2 in DTT treated smooth muscle cells. TG2 substrates
were labeled with BPA or Q-peptide and subsequently purified by
streptavidin pull down. Control cells were treated with DTT only.
(A) Coomassie staining of the total lysates and pull downs
(* indicates streptavidin band). (B) Western blot stained with
antibodies against fibronectin and nidogen-1 shows specific
labeling of both proteins with the lysine donor BPA. For
fibronectin an additional high molecular weight product can be
Fibronectin and nidogen-1 are substrates for
observed which is labeled with both the lysine and the glutamine
with DTT did not significantly alter the vessel response to the
thromboxane analogue U46619. After incubating vessels for
24 hrs with 2 mmol/L DTT, maximal contraction to U46619
was unchanged. Data are expressed as percentage of vessel
diameter at full relaxation.
Effect of DTT on vessel reactivity. Treatment
Exogenous, recombinant TG2 required a reducing agent to
induce inward remodeling, which was accomplished by cross-
linking. Only a cell-permeable reducing agent activates a pool of
endogenous TG2 that can induce inward remodeling. Intracellular
TG2 translocates to the cell surface and subsequently cross-links a
number of proteins. This can be prevented with a site-specific
inhibitor of TG2 or an NO donor. Exposure to calcium ionophore
increases intracellular transglutaminase activity, which also can be
counteracted with SNAP. In this case, however, inward remod-
eling is absent.
Schematic overview of experimental results.
visualized by incorporation of AlexaFluor594/cadaverine and
scanned by confocal microscopy over different layers of the blood
vessel. TG2 activity was low in the intima and adventitia. Smooth
muscle cells in the medial layer, aligned perpendicular to the vessel
long axis, display strong membrane-bound TG2 activity. In
addition, patches of transglutaminase activity were observed
between the endothelium and smooth muscle layer.
TG2 activity in the vessel wall. TG2 activity was
Technical assistance by Judith de Vos is gratefully acknowledged.
Conceived and designed the experiments: JvdA EVB EB. Performed the
experiments: JvdA RvG HLM BGT GMCJ PAvV EB. Analyzed the data:
JvdA EVB RvG WCB GMCJ PAvV JGRDM EB. Wrote the paper: JvdA
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