Basic & Clinical Pharmacology & Toxicology, 108, 318–325 Doi: 10.1111/j.1742-7843.2010.00656.

Doxycycline Dose-dependently Inhibits MMP-2-Mediated Vascular

Changes in 2K1C Hypertension
Danielle A. Guimaraes1, Elen Rizzi1, Carla S. Ceron1, Alisson M. Oliveira1, Diogo M. Oliveira1, Michele M. Castro1, Carlos R. Tirapelli2,
Raquel F. Gerlach3 and Jose E. Tanus-Santos1
1
Department of Pharmacology, Faculty of Medicine of Ribeir¼o Preto, University of S¼o Paulo (USP), 2Department of Psychiatric Nursing
and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeir¼o Preto, University of S¼o Paulo (USP), and 3Department of
Morphology, Estomatology and Physiology, Dental School of Ribeir¼o Preto, University of S¼o Paulo, Ribeir¼o Preto, SP, Brazil
(Received 16 August 2010; Accepted 4 November 2010)

Abstract: Hypertension induces vascular alterations that are associated with up-regulation of matrix metalloproteinases
(MMPs). While these alterations may be blunted by doxycycline, a non-selective MMPs inhibitor, no previous study has
examined the effects of different doses of doxycycline on these alterations. This is important because doxycycline has been
used at sub-antimicrobial doses, and the use of lower doses may prevent the emergence of antibiotic-resistant microorganisms.
We studied the effects of doxycycline at 3, 10 and 30 mg ⁄ kg per day on the vascular alterations found in the rat two kidney-
one clip (2K1C) hypertension (n = 20 rats ⁄ group). Systolic blood pressure (SBP) was monitored during 4 weeks of treatment.
We assessed endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aor-
tic wall was studied, and aortic MMP-2 levels ⁄ proteolytic activity were determined by gelatin and in situ zymography, respec-
tively. All treatments attenuated the increases in SBP in hypertensive rats (195.4 € 3.9 versus 177.2 € 6.2, 176.3 € 4.5, and
173 € 5.1 mmHg in 2K1C hypertensive rats treated with vehicle, or doxycycline at 3, 10, 30 mg ⁄ kg per day, respectively (all
p < 0.01). However, only the highest dose prevented 2K1C-induced reduction in endothelium-dependent vasorelaxation
(p < 0.05), vascular hypertrophy and increases in MMP-2 levels (all p < 0.05). In conclusion, our results suggest that relatively
lower doses of doxycycline do not attenuate the vascular alterations found in the 2K1C hypertension model, and only the
highest dose of doxycycline affects MMPs and vascular structure. Our results support the idea that the effects of doxycycline
on MMP-2 and vascular structure are pressure independent.

Hypertension is a major cardiovascular disease that is associ-
ated with vascular remodelling characterized by degradation
and reorganization of extracellular matrix in the vessel wall
[1]. Vascular remodelling is an adaptive response to elevation
of arterial pressure to normalize the wall tension [2] and has
been clearly described in experimental models of hyperten-
sion, including the 2-kidney, 1-clip (2K1C) Goldblatt model,
which involves the activation of the renin–angiotensin–aldo-
sterone axis [3,4].
Metalloproteinases (MMPs) are zinc-containing enzymes
that play important roles in cardiovascular diseases [5]. Up-
regulated MMPs promote excessive degradation of extracel-
lular matrix and are involved in pathological vascular
remodelling [6], which includes vascular smooth muscle cell
migration and proliferation in the arterial wall [7]. In fact,
increased expression and activity of MMP-2 (gelatinase A)
and MMP-9 (gelatinase B) have been consistently implicated
in vascular remodelling associated with hypertension in
patients [8,9] and animal models [10–16]. We have recently
found evidence suggesting that enhanced aortic MMP-2 lev-
els and activity may underlie the impaired endothelial-depen-
Author for correspondence: Jose Eduardo Tanus-Santos, Depart-
ment of Pharmacology, Faculty of Medicine of Ribeir¼o Preto, Uni-
versity of S¼o Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeir¼o
Preto, SP, Brazil (fax +55 16 3633 2301, e-mails tanus@fmrp.usp.br;
tanussantos@yahoo.com).
dent vasorelaxation, arterial wall hypertrophy, and excessive
collagen and elastin deposition in 2K1C hypertension,
thereby contributing to hypertensive vascular remodelling
and dysfunction [15].
The contribution of MMPs to disease conditions justified
the search for MMP inhibitors. Interestingly, doxycycline (a
tetracycline) was shown to inhibit MMP activity at plasma
concentrations below those required to produce antimicro-
bial effects and is one of the most potent non-selective MMP
inhibitors [17,18]. In fact, several studies have shown benefi-
cial effects of doxycycline in many disease models [19–25].
However, no previous study has comparatively examined the
effects of different doses of doxycycline on the vascular alter-
ations found in 2K1C hypertensive rats. Examining the
effects of different doses of doxycycline is important because
this drug has been used at sub-antimicrobial doses, and the
use of lower doses may prevent the emergence of antibiotic-
resistant microorganisms. In this study, we compared the
effects of three doses of doxycycline (3, 10 and 30 mg ⁄ kg) on
the vascular changes associated with 2K1C hypertension.
Materials and Methods
Animals and treatments. This study complied with the guidelines of
the Faculty of Medicine of Ribeir¼o Preto, University of S¼o Paulo,
and the animals were handled according to the guiding principles
published by the National Institutes of Health Guide for the Care

 2010 The Authors

Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society
 DOXYCYCLINE INHIBITS HYPERTENSION
and Use of Laboratory Animals. Male Wistar rats (180–200 g)
obtained from the colony at University of S¼o Paulo were main-
tained in a 12-hr light : dark cycle at 25C with free access to rat
chow and water.
2K1C hypertension was induced by clipping the left renal artery
with a silver clip (0.2 mm). Sham-operated rats underwent the same
surgical procedure (under general anaesthesia with ketamine
100 mg ⁄ kg and xylazine 10 mg ⁄ kg i.p.), except for the placement of
the renal artery clip. The animals were randomly assigned to one of
eight groups: 2K1C and sham groups that received tap water; 2K1C
and Sham groups that received doxycycline at 3 mg ⁄ kg, a non-selec-
tive MMP inhibitor; 2K1C and Sham groups that received doxycy-
cline at 10 mg ⁄ kg; and 2K1C and Sham groups that received
doxycycline at 30 mg ⁄ kg. Doxycycline was given by gavage, and dis-
tilled water was used as vehicle. Treatment with doxycycline was
started 2 weeks after 2K-1C hypertension was induced and main-
tained for an additional 4 weeks. Body-weight and tail systolic blood
pressure were assessed weekly throughout the experiment period.
The systolic blood pressure was measured by tail-cuff plethysmogra-
phy in conscious rats.
Vascular reactivity. After 4 weeks of treatment with doxycycline (or
vehicle), the animals were killed by decapitation, and their thoracic
aortas were isolated and cleaned of connective tissue and fat. Aortic
rings, 4 mm in length, were cut and mounted for isometric tension
recording [26]. The rings were placed in bath chambers (5 mL) for
isolated organs containing modified Krebs salt solution (KSS) with
the following composition (mM): NaCl 130, CaCl2 1.6, MgSO4 1.2,
KH2PO4 1.2, KCl 4.7, NaHCO3 14.9, glucose 5.5, which was main-
tained at 37C, pH 7.4 and bubbled with 95% O2 and 5% CO2. The
system was connected to an isometric force displacement transducer
(Letica Scientific Instruments; Barcelona, Spain), and the responses
were recorded on a computer system using the Chart V4.04, Power-
Lab ADInstruments (2000) Program. The aortic rings were submit-
ted to a basal tension of 1.5 g during a 60-min. equilibration period
and were considered to have an intact functional endothelium when
acetylcholine (10)6 M) produced a relaxation of more than 80%.
Relaxation was calculated as a percentage of the contraction induced
by phenylephrine (10)7 M). To assess endothelium-dependent and
endothelium-independent relaxations, aortic rings pre-contracted
with phenylephrine (10)7 M) were used to construct cumulative con-
centration–response curves to acetylcholine (10)10–10)5 M) and
sodium nitroprusside (10)10–10)6 M), respectively. Concentration–
response curves were fitted using a non-linear interactive fitting pro-
gram (Graph Pad Prism 5.0; GraphPad Software Inc., La Jolla, CA,
USA). Agonist potencies were expressed as pD2 (negative logarithm
of the molar concentration of agonist producing 50% of the maxi-
mum response), and maximum response was expressed as Emax
(maximum effect elicited by the agonist).
Morphometric analysis of the vascular wall. After 4 weeks of treat-
ment with doxycycline (or vehicle), the rats were killed by decapita-
tion. Their thoracic aorta were harvested, cleaned off connective
tissue and immediately fixed in 4% phosphate-buffered paraformal-
dehyde, pH 7.4, and embedded in paraffin blocks. Four-lm-thick
slices were stained with haematoxylin and eosin (H&E). Media
cross-sectional area (CSA) was calculated by subtracting the lumen
internal area (Ai) from the external area (Ae), which were measured
in tissue sections (250·) [27]. The external diameter (ED) and the
internal diameter (ID) were calculated as the square root of 4Ae ⁄ p
and 4Ai ⁄ p, respectively. Media thickness (M) was calculated as (ED-
ID) ⁄ 2. Finally, M to lumen diameter (M ⁄ L) was also calculated.
The number of vascular smooth muscle cells in the aortic wall was
measured by the tridimensional disector method on two consecutive
sections, as previously described [27]. This method is independent of
nuclei orientation, form and size. Typical nuclei from smooth muscle
cells are about 5–8 microns wide; therefore, they are large enough
not to be completely enclosed by one section. Stained sections were
examined with light microscopy (DMLB; Leica, Bensheim, Ger-
many), and the image was captured at ·400, as previously described
 2010 The Authors
Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 108, 318–325
319
[27]. These structural analyses in the media were evaluated by using
ImageJ Program [National Institute of Health (NIH)].
Measurement of Aortic MMP-2 Levels by Gelatin Zymography. Tis-
sues were homogenized in buffer containing 20 mmol ⁄ l Tris–HCl,
pH 7.4, 1 mmol ⁄ l 1,10-phenanthroline and 1 mmol ⁄ l PMSF, 1 mM
NEM, and 10 mM CaCl2. Briefly, tissue extracts normalized for
protein concentration were subjected to electrophoresis on 12%
SDS–PAGE copolymerized with gelatin (1%) as the substrate. After
electrophoresis, the gel was incubated for 1 hr at room temperature
in a 2% Triton X-100 solution, washed two times with water and
incubated at 37C for 16 hr in Tris–HCl buffer, pH 7.4, containing
10 mmol ⁄ l CaCl2. The gels were fixed with 30% methanol and 10%
acetic acid, stained with 0.05% Coomassie Brilliant Blue G-250 and
then destained with 30% methanol and 10% acetic acid. Gelatinolytic
activities were detected as unstained bands against the background
of Coomassie blue-stained gelatin, assayed by densitometry using the
ImageJ Program [National Institute of Health (NIH)]. Intergel
analysis was possible after normalization of gelatinolytic activity with
an internal standard (culture medium conditioned by fibroblasts).
The forms of MMP-2 were identified as bands at 75, 72 and 64 KDa
[28–30], which were inhibited by phenanthroline and not by other
proteinase inhibitors, and were further identified by immunoprecipi-
tation with specific antibodies. Drugs and reagents were purchased
from Sigma-Aldrich (St. Louis, MO, USA).
In situ zymography and immunofluorescence for MMP-2.
MMP activity in the media and intima of thoracic aorta was mea-
sured using DQ Gelatin (E12055, Molecular Probes, Eugene, OR,
USA) as the fluorogenic substrate. Briefly, aortic tissues were
embedded vertically in Tissue-tek and were frozen and cut in serial
4-lm sections. Vessel sections were incubated in dark humidified
chambers for 1 hr with DQ gelatin at a concentration 1.0 lg ⁄ ml in
Tris-CaCl2 Buffer (Tris at 50 mM, CaCl2 at 10 mM, ZnCl2 at
1 lM). The sections were examined with fluorescent microscopy
(Leica Imaging Systems Ltd., Cambridge, UK), and the image was
captured at ·400. Proteolytic activity was detected as bright green
fluorescence, indicating substrate breakdown and MMP enzymatic
activity, and was evaluated by using ImageJ Program [National
Institute of Health (NIH)]. Twenty arbitrary fields, with predeter-
mined area, were selected around the vessel circumference for each
section from each animal (n = 4–5). The arithmetic mean of the flu-
orescence from the 20 fields was calculated for each slide. This
number of fields per slide corresponds to approximately 20–30% of
the total aortic area being studied and has led to interassay coeffi-
cients of variations of less than 3%. Phenantroline 1 mM and
PMSF 1 mM were used to confirm the activity of MMPs in tho-
racic aorta. While phenantroline fully inhibited MMP activity,
PMSF did not do the degradation of the substrate (data not
shown).
Aortic MMPs activity was also colocalized with aortic MMP-2
expression by immunofluorescence. After DQ gelatin, tissue sections
were incubated with MMP-2 primary mouse anti-human monoclo-
nal antibody (MAB3308; Chemicon, Billerica, MA, USA), for 1 hr
in dark humidified chambers [MMP-2 (1:1000)]. Red fluorescence
was visualized by adding the rhodamine conjugated as secondary
antibody (1:200) (AP160P, Chemicon) for 1 hr. To confirm the
specificity of antibodies, the primary antibody was omitted and
substituted with PBS + 1% BSA. Rhodamine did not bind non-
specifically to tissue sections. Photoshop software was used to
colocalize the aortic photographs.
In vitro effects of doxycycline on MMP-2 activity. To examine
whether doxycycline (from 0.8 to 200 lM) directly inhibits MMP-2
activity in vitro, we measured human recombinant MMP-2 activity
(R&D Systems Inc., Minneapolis, MN, USA) in absence or presence
of doxycycline by using the Gelatinolytic Activity Kit (E12055,
Molecular Probes). This activity was evaluated in a microplate spec-
trofluorometer (at kexcitation 495, kemission 515 nm; Gemini EM,
Molecular Devices, Sunnyvale, CA, USA) after 30 min. of incuba-
320 DANIELLE A. GUIMARAES ET AL.

tion at 37C, as previously described [31]. Phenanthroline (0.1 mM)
was used as a positive control for MMP-2 activity inhibition.
Statistical analysis. Results are expressed as means € S.E.M.
Between-group comparisons were assessed by two-way or one-way
ANOVA followed by the Tukey test using GraphPad Prism software. A
probability value <0.05 was considered significant.
Results
Effect of different doses of doxycycline on systolic blood
pressure levels and body-weight in 2K1C rats.
Baseline systolic blood pressure and body-weight were simi-
lar in the eight experimental groups (fig. 1A and B; n = 20
rats ⁄ group). After the first week of the renal artery surgery
in the 2K1C group, systolic blood pressure increased pro-
gressively until the sixth week of study (fig. 1A). All doses of
doxycycline attenuated the increases in systolic blood pres-
sure in hypertensive rats after the fifth week of the renal
artery surgery (195.4 € 3.9 versus 177.2 € 6.2, 176.3 € 4.5
and 173 € 5.1 mmHg in 2K1C and 2K1C+doxy 3,
2K1C+doxy 10 and 2K1C+doxy 30 groups, respectively, at
the sixth week; all p < 0.01; fig. 1A). No significant changes
in systolic blood pressure were seen in sham and sham +
doxycycline rats during the 6 weeks of treatment (fig. 1A).
No major differences in body-weight were observed among
all the experimental groups (fig. 1B).
Treatment
A
or
Vehicle
Systolic blood pressure (mmHg)
Effect of different doses of doxycycline on vascular function
and remodelling in 2K1C rats.
To evaluate the effects of different doses of doxycycline
on vascular function, rat aortic rings were isolated, and
their functional responses to acetylcholine and sodium
nitroprusside (n = 5 per group) were assessed in organ
chamber experiments. Fig. 2 shows endothelial cell-depen-
dent vasorelaxation induced by acetylcholine (Ach; 10)10–
10)5 M). An impaired response to Ach was observed in
the 2K1C group (p < 0.05; fig. 2). Only the treatment
with doxycycline at 30 mg ⁄ kg reversed the 2K1C-induced
impairment in vascular relaxation to Ach (p < 0.05;
fig. 2). While no significant differences were observed in
pD2 obtained in response to Ach, 2K1C hypertension
reduced the maximum effect by approximately 25%
(p < 0.01; fig. 2B), and this reduction was blunted only
by the treatment with doxycycline at 30 mg ⁄ kg (fig. 2B).
We found no significant alterations in the response to
sodium nitroprusside in all experimental groups (data not
shown). Vascular reactivity to Ach and sodium nitroprus-
side was not affected by any treatment in the sham
groups.
Renovascular hypertension was associated with arterial
wall hypertrophy, with significant increases in the number of
vascular smooth muscle cells and with increased aortic CSA
and M ⁄ L ratio (fig. 3A and B; all p < 0.05). Only treatment
with doxycycline at 30 mg ⁄ Kg attenuated all these structural
alterations associated with hypertension (fig. 3A and B; all
p < 0.05). Doxycycline at 10 and 30 mg ⁄ kg prevented
2K1C-induced increases in M ⁄ L (fig. 3A and B; p < 0.05).
No structural difference was found in the aortas from rats in

210 the sham and sham + doxycycline groups (p > 0.05).

* Sham
200 *
190 * * * Sham + doxy 3
180 * ** ** Effect of different doses of doxycycline on the levels and
Sham + doxy 10
170
Sham + doxy 30 activity of MMP-2 in 2K1C rats.
160
150 2K1C Gelatin zymography was used to evaluate aortic MMP levels
140 2K1C + doxy 3 in all experimental groups. Fig. 4A shows a zymogram gel
130 2K1C + doxy 10
120 with bands corresponding to molecular weights of the
2K1C + doxy 30
110 MMP-2 bands (75, 72 and 64 kDa). Aortas from 2K1C rats
100
0 1 2 3 4 5 6 7 showed higher levels of the three bands of MMP-2 when
Time (Weeks) compared with the sham groups (n = 7–12 per group;
p < 0.05; fig. 4B). Treatment with doxycycline at 30 mg ⁄ kg,
B 600 but not at 3 or 10 mg ⁄ kg, attenuated 2K1C-induced
500 Treatment
increases in 75, 64 kDa and total MMP-2 levels (p < 0.05;
Body weight (g)

or fig. 4B).
400 Vehicle
A semiquantitative determination of the gelatinolytic
300
activity was carried out in the aortas by in situ zymography
200 (n = 6–8 per group). We found increased green fluorescence
100 in the endothelium and media of thoracic aorta from
0
2K1C rats compared with the sham groups (p < 0.05;
0 1 2 3 4 5 6 7 fig. 5A and B). Treatment with doxycycline at 30 mg ⁄ kg
Time (Weeks) attenuated 2K1C-induced increases in total MMP activity
(p < 0.05; fig. 5A and B). The increased gelatinolytic activ-
Fig. 1. Systolic blood pressure (mmHg) measured by tail-cuff
ity was colocalized with increased aortic MMP-2 expression
method (panel A), and body-weight (panel B) in the eight experi-
mental groups during the study period. Data are shown as mean € - detected by immunofluorescence (n = 6–8 per group) in
S.E.M.; (n = 20 per group). *p < 0.001 versus the sham group; 2K1C rats compared with those found in the sham groups
**p < 0.01 versus the 2K1C group. (p < 0.05; fig. 5A and C). In parallel with the in situ

 2010 The Authors

Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 108, 318–325
 DOXYCYCLINE INHIBITS HYPERTENSION 321

Sham
0 2K1C
A B
2K1C+doxy 3
20 2K1C+doxy 10
150 10.0

Vascular relaxation (%)

2K1C+doxy 30

pD 2 (–log EC50) Ach

40 *

Emax (%) Ach

* 7.5
100

60 5.0

50
80 2.5

* 0 0.0
100

2 m
1C do C
1C dox 3
ox 0
30

2 m
1C do C
1C dox 3
ox 0
30
+d y 1

+d y 1
2K 1C+ K1

2K 1C+ K 1
a

2K + xy

2K + xy
Sh

Sh

y
*
* *
120

2K

2K
–10 –9 –8 –7 –6 –5
Ach (log (M))

Fig. 2. Endothelial cell-dependent vasorelaxation (panel A), maximum relaxation (Emax %) and pD2 (panel B) induced by Ach in rat aortic
rings preparations. Vascular responses were studied after pre-constriction with phenylephrine (10)4 M). Data are shown as mean € S.E.M.
(n = 5 per group). *p < 0.05 versus the 2K1C group.

A Vehicle Doxy 3 Doxy 10 Doxy 30

Sham

2K1C

B 50 20
200,000 *
* *
40
Cells/μm aorta

** **
CSA (μm2)

**
M/L (%)

30
100,000 10
20 **
10

0 0 0
30
10
3
1C
am

am

1C

10

30
am

1C

10

30
xy

y
xy
2K

y
xy

2K
2K

y
xy

xy
Sh

Sh
Sh
x
do

do

x
do
do
do

do

do
do

do
+

+
+
+
+

+
+

+
1C

1C
1C
1C
1C

1C

1C
1C

1C
2K

2K
2K
2K
2K

2K

2K
2K

2K

Fig. 3. Effects of doxycycline on aortic structural modifications induced by 2K1C hypertension. Panel A shows representative photographs of
aortic samples (400·) stained by hematoxylin and eosin (H&E). Panel B shows the values for vascular smooth muscle cells number in the aortic
media, medial cross-sectional area (CSA) and media to lumen ratio (M ⁄ L). Data are shown as mean € S.E.M. (n = 5-8 per group). * p < 0.05
versus the sham group. ** p < 0.05 versus the 2K1C group.

zymography results, we found lower red fluorescence (lower In vitro effects of doxycycline on MMP-2 activity.
MMP-2 levels detected by immunofluorescence) in the We found that doxycycline significantly inhibited human rec-
2K1C+doxy 30 group compared with the 2K1C group ombinant MMP-2 activity when present at 100 or 200 lM
(p < 0.05; fig. 5A and C). concentrations (both p < 0.05; fig. 6; n = 3 per concentra-

 2010 The Authors

Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 108, 318–325
322 DANIELLE A. GUIMARAES ET AL.

A
←
←
75 KDa
72 KDa
← 64 KDa

am

1C

10

do 10
30

30
ST

xy

xy
2K

xy

y
xy

xy
Sh

do

do

am dox
do

do
+

+
am

1C

am

1C

1C
2K
Sh

2K

2K
Sh

Sh
B 4

(arbitrary units)
MMP-2, 72 kDa
(arbitrary units) 1.5 3 *
MMP-2, 75 kDa

1.0
* 2
**
0.5 1

0.0 0
am

1C

10

30

am

1C

10

30
xy

xy
2K

2K
xy

xy

xy

xy
Sh

Sh
do

do
do

do

do

do
+

+
+

+
1C

1C
1C

1C

1C

1C
2K

2K
2K

2K

2K

2K
0.75 5
*

MMP-2 total levels

(arbitrary units)
64 kDa MMP-2

(arbitrary units)
4
0.50
**
3
**
2
0.25
1

0.00 0
am

1C

10

30
am

1C

10

30

xy
xy

2K
2K

xy

xy
xy

xy

Sh
Sh

do
do

do

do
do

do

+
+

+
+

1C
1C

1C

1C
1C

1C

2K
2K

2K

2K
2K

2K

Fig. 4. Panel A shows a representative SDS–PAGE gelatin zymogram of aortic samples. Molecular weights of MMP-2 bands were identified
after electrophoresis on 12% SDS–PAGE. STD: internal standard. Panel B shows the values for each molecular weight form (75, 72, 64 kDa
and total MMP-2 levels) in the aortas. Data are shown as mean € S.E.M. (n = 7–12 per group). *p < 0.05 versus the sham group. **p < 0.05
versus the 2K1C group.

tion). Doxycycline at concentrations below 50 lM produced
no significant effects on human recombinant MMP-2 activ-
ity (p > 0.05; fig. 6), although phenanthroline 100 lM pro-
duced almost complete inhibition (fig. 6; p < 0.05).
Discussion
The main finding of the present study was that treatment
with doxycycline at 30, but not 3 or 10 mg ⁄ kg per day,
decreased MMPs levels and gelatinolytic activity and
blunted the vascular alterations found in 2K1C hyperten-
sive rats. This is the first study to compare the effects of
significantly different doses of doxycycline on biochemical,
morphological and functional alterations caused by 2K1C
hypertension.
Doxycycline inhibits MMP expression and activity and
has been used in many disease models (including hyperten-
sion) to assess the possible role of MMPs in cardiovascular
diseases [15,16,26]. However, there are discrepancies in the
literature related to the effects of doxycycline on cardiovas-
cular diseases, which could be explained by differences in the
dose used in these studies. For example, while oral treatment
with doxycycline at 30 mg ⁄ kg per day improved post-myo-
cardial infarction (MI) remodelling and left ventricular func-
tion in a rat MI model and reduced infarct size in a
myocardial ischaemia-reperfusion model [32,33], doxycycline
at 160 mg ⁄ kg per day accelerated the development of cardiac
hypertrophy and the progression to heart failure after aortic
constriction [34]. Moreover, a relatively low dose of doxycy-
cline (15 mg ⁄ kg per day) reversed mechanical, electrical and
biochemical alterations found in the hearts from diabetic rats
[19]. To our knowledge, there are no previous studies com-
paring the effects of different doses of doxycycline on the
vascular alterations associated with hypertension. Addressing
this issue may be important because previous studies showed
variable effects produced by different doses of doxycycline.
Although we have recently shown that doxycycline at
30 mg ⁄ kg per day completely reverted the structural and
functional vascular changes associated with 2K1C hyperten-
sive rats [15], there is no experimental evidence that lower
doses would produce similar effects.
The morphological and functional vascular alterations
that we found in 2K1C are in agreement with previous
reports [10,15,27]. Treatment with doxycycline 30 mg ⁄ kg per
day completely reversed the morphological and functional
alterations found in 2K1C rats, thus confirming previous

 2010 The Authors

Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 108, 318–325
 DOXYCYCLINE INHIBITS HYPERTENSION 323

A Vehicle Doxy 3 Doxy 10 Doxy 30 Vehicle Doxy 3 Doxy 10 Doxy 30

Gelatinase
activity 200 μm 200 μm

MMP-2
200 μm
200 μm

Merge
200 μm 200 μm

Sham 2K1C

B
In situ gelatinolityc activity

40 C 15

* *

(arbitrary units)
(arbitrary units)

MMP-2 levels
30
10 **
20
**

5
10

0 0
am

1C

10

30

am

1C

10

30
xy

ox
2K

2K
xy

xy

ox

ox
Sh

Sh
do

D
do

do

D
+
+

+
1C
+

+
1C

1C

1C
1C

1C

2K
2K

2K

2K
2K

2K

Fig. 5. Effects of doxycycline on in situ gelatinase activity and MMP-2 levels by immunofluorescence in the aortas from hypertensive rats. Panel
A shows representative photographs of gelatinase activity (·400), MMP-2 detected by immunofluorescence, and their colocalization in vessels
surface. Panel B shows the quantification of bright green fluorescence, which reflects gelatinolytic activity. Panel C shows the quantification of
red fluorescence, which reflects MMP-2 level. Data are shown as mean € S.E.M. (n = 6-8 per group). *p < 0.05 versus the sham group.
**p < 0.05 versus the 2K1C group.

4000
3500
Fluorescent intensity

3000
(arbitrary units)

2500
2000
1500 *
1000
500
* * *
0
e

50

25

.5

8
cl

6.

3.

1.

0.
Ph

20

10

12
hi
Ve

Doxycycline

Fig. 6. Effect of doxycycline at different concentrations (from 0.8 to 200 lM) on the activity of recombinant MMP-2 (rMMP-2). Phenanthro-
line (Phe) 0.1 mM was used as a positive control for MMP-2 inhibition. Data are shown as mean € S.E.M. (n = 3). *p < 0.05 versus vehicle.

findings [15]. However, although the lower doses (3 and lower doses of doxycycline produced minor (if any) MMP
10 mg ⁄ kg per day) produced antihypertensive effects that inhibition.
were similar to those associated with the highest dose Our results clearly show that the effects of doxycycline on
(30 mg ⁄ kg per day), treatment with 10 mg ⁄ kg per day pro- MMP-2 and vascular structure are pressure independent
duced only slight, non-significant effects on the structural because all doses of doxycycline produced the same reduc-
and functional changes associated with 2K1C hypertension. tion in arterial blood pressure. Although unproven, we do
In fact, our biochemical (gel zymography, in situ zymography not believe that the normalization of structure, Ach relaxa-
and immunofluorescence for MMP-2) confirmed that the tion and MMP-2 level to the level of sham-operated animals

 2010 The Authors

Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 108, 318–325
324 DANIELLE A. GUIMARAES ET AL.
are because of the small reduction in BP. Conversely, we
really believe that the normalization of these alterations asso-
ciated with hypertension is because of MMPs inhibition by
doxycycline, as previously shown [15].
Our previous studies performed with this hypertension
model show that blood pressure peaks between 4 and
6 weeks after hypertension is induced [15,16,31,35]. This ani-
mal model of hypertension is clearly associated with
increased angiotensin II levels, especially during the first
4 weeks. The lack of significant effects for the high dose of
doxycycline during the first 2 weeks of treatment is highly
suggestive that MMPs do not play a major role in hyperten-
sion at this initial phase, and that time-dependent changes in
MMPs alterations may be relevant in this hypertension
model. However, this hypothesis remains to be proved.
An obvious reason for lack of effect for the lower doses of
doxycycline is the lack of sufficient concentrations of this
MMP inhibitor in the vascular tissue. In this regard, Prall
et al. [36] showed that treatment with doxycycline at 10, 50
and 100 mg ⁄ kg reduced abdominal aortic aneurysmal
growth in mice and produced dose-dependent increases with
the mean serum doxycycline concentrations achieving 1.4,
2.7 and 11.9 lg ⁄ ml, respectively [36], which correspond to
2.7, 5.3 and 23.2 lM. If we assume that the doxycycline con-
centrations achieved in rats from the present study were pro-
portional to those reported by Prall et al. [36], we would
expect to find doxycycline concentrations varying from 1 to
<5 lM in the present study. Indeed, Prall et al. [36] sug-
gested that doxycycline at concentrations >4 lM is likely
effective in reducing MMPs concentrations, and this sugges-
tion supports our findings showing no significant effects for
doxycycline at 10 mg ⁄ kg per day or less. In addition, while
our in vitro experiments showed that doxycycline at concen-
trations below 50 lM produced no significant effects on
human recombinant MMP-2 activity, these in vitro studies
may not reflect the in vivo effects produced by chronic treat-
ment with doxycycline [17,18], including the reduction in
MMP mRNA stability, thus down-regulating MMP expres-
sion [22].
The highest dose of doxycycline tested in the present study
is significantly higher than the dose used in human beings.
The mean circulating concentrations reached after a single
oral 100 mg dose to adults is approximately 2 lg ⁄ ml or
4 lM [37]. If we could extrapolate our results to clinical
hypertension, our results could be consistent with the idea
that clinical doses of doxycycline may normalize the abnor-
mal vasculature in essential hypertension. This suggestion is
highly speculative, and a clinical trial examining the effects
of doxycycline in hypertensive patients is required.
Finally, because we found that doxycycline at lower doses
does not protect from vascular damage in 2K1C hyperten-
sion, whereas 30 mg ⁄ kg does, it would be interesting to
examine the effects produced by doses higher than
30 mg ⁄ kg. In fact, we tested the effects produced by
100 mg ⁄ kg (data not shown). We found that many animals
died after 100 mg ⁄ kg daily, thus suggesting that the thera-
peutic window for doxycycline may be rather narrow. There-
 2010 The Authors
Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 108, 318–325
fore, we decided to stop studying the effects associated with
this dose.
In conclusion, our results suggest that relatively lower
doses of doxycycline do not attenuate the vascular altera-
tions found in the 2K1C hypertension model, and only the
highest dose of doxycycline affects MMPs and vascular
structure. Our results support the idea that the effects of
doxycycline on MMP-2 and vascular structure are blood
pressure independent.
Acknowledgements
This study was funded by Fundażo de Amparo a Pes-
quisa do Estado de S¼o Paulo (FAPESP-Brazil) and Conse-
lho Nacional de Desenvolvimento Cientfico e Tecnolgico
(CNPq-Brazil).
References
1 Raffetto JD, Khalil RA. Matrix metalloproteinases and their
inhibitors in vascular remodeling and vascular disease. Biochem
Pharmacol 2008;75:346–59.
2 Safar ME, London GM, Asmar R, Frohlich ED. Recent
advances on large arteries in hypertension. Hypertension
1998;32:156–61.
3 Lerman LO, Chade AR. Atherosclerotic process, renovascular
disease and outcomes from bench to bedside. Curr Opin Nephrol
Hypertens 2006;15:583–7.
4 Textor SC. Renovascular hypertension update. Curr Hypertens
Rep 2006;8:521–7.
5 Schulz R. Intracellular targets of matrix metalloproteinase-2 in
cardiac disease: rationale and therapeutic approaches. Annu Rev
Pharmacol Toxicol 2007;47:211–42.
6 Sluijter JP, de Kleijn DP, Pasterkamp G. Vascular remodeling
and protease inhibition–bench to bedside. Cardiovasc Res
2006;69:595–603.
7 Newby AC. Matrix metalloproteinases regulate migration, prolif-
eration, and death of vascular smooth muscle cells by degrading
matrix and non-matrix substrates. Cardiovasc Res 2006;69:614–24.
8 Yasmin SW, McEniery CM, Dakham Z, Pulsalkar P, Maki-
Petaja K, Ashby MJ et al. Matrix metalloproteinase-9 (MMP-9),
MMP-2, and serum elastase activity are associated with systolic
hypertension and arterial stiffness. Arterioscler Thromb Vasc Biol
2005;25:372.
9 Ahmed SH, Clark LL, Pennington WR, Webb CS, Bonnema
DD, Leonardi AH et al. Matrix metalloproteinases ⁄ tissue inhibi-
tors of metalloproteinases: relationship between changes in prote-
olytic determinants of matrix composition and structural,
functional, and clinical manifestations of hypertensive heart dis-
ease. Circulation 2006;113:2089–96.
10 Bouvet C, Gilbert LA, Girardot D, deBlois D, Moreau P. Differ-
ent involvement of extracellular matrix components in small and
large arteries during chronic NO synthase inhibition. Hyperten-
sion 2005;45:432–7.
11 Watts SW, Rondelli C, Thakali K, Li X, Uhal B, Pervaiz MH
et al. Morphological and biochemical characterization of remod-
eling in aorta and vena cava of DOCA-salt hypertensive rats. Am
J Physiol Heart Circ Physiol 2007;292:H2438–48.
12 Lehoux S, Lemarie CA, Esposito B, Lijnen HR, Tedgui A. Pres-
sure-induced matrix metalloproteinase-9 contributes to early
hypertensive remodeling. Circulation 2004;109:1041–7.
13 Flamant M, Placier S, Dubroca C, Esposito B, Lopes I, Chatzian-
toniou C et al. Role of matrix metalloproteinases in early hyper-
tensive vascular remodeling. Hypertension 2007;50:212–8.
 DOXYCYCLINE INHIBITS HYPERTENSION
14 Martinez ML, Castro MM, Rizzi E, Fernandes K, Demacq C,
Bendhack LM et al. Lercanidipine reduces matrix metalloprotein-
ase-2 activity and reverses vascular dysfunction in renovascular
hypertensive rats. Eur J Pharmacol 2008;591:224–30.
15 Castro MM, Rizzi E, Figueiredo-Lopes L, Fernandes K,
Bendhack LM, Pitol DL et al. Metalloproteinase inhibition
ameliorates hypertension and prevents vascular dysfunction
and remodeling in renovascular hypertensive rats. Atherosclero-
sis 2008;198:320–31.
16 Castro MM, Rizzi E, Prado CM, Rossi MA, Tanus-Santos JE,
Gerlach RF. Imbalance between matrix metalloproteinases and
tissue inhibitor of metalloproteinases in hypertensive vascular
remodeling. Matrix Biol 2010;29:194–201.
17 Lee HM, Ciancio SG, Tuter G, Ryan ME, Komaroff E, Golub
LM. Subantimicrobial dose doxycycline efficacy as a matrix me-
talloproteinase inhibitor in chronic periodontitis patients is
enhanced when combined with a non-steroidal anti-inflammatory
drug. J Periodontol 2004;75:453–63.
18 Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J,
Sorsa T. Tetracyclines inhibit connective tissue breakdown by
multiple non-antimicrobial mechanisms. Adv Dent Res 1998;12:-
12–26.
19 Yaras N, Sariahmetoglu M, Bilginoglu A, Aydemir-Koksoy A,
Onay-Besikci A, Turan B et al. Protective action of doxycycline
against diabetic cardiomyopathy in rats. Br J Pharmacol
2008;155:1174–84.
20 Wang W, Schulze CJ, Suarez-Pinzon WL, Dyck JR, Sawicki G,
Schulz R. Intracellular action of matrix metalloproteinase-2
accounts for acute myocardial ischemia and reperfusion injury.
Circulation 2002;106:1543–9.
21 Tessone A, Feinberg MS, Barbash IM, Reich R, Holbova R,
Richmann M et al. Effect of matrix metalloproteinase inhibition
by doxycycline on myocardial healing and remodeling after myo-
cardial infarction. Cardiovasc Drugs Ther 2005;19:383–90.
22 Liu J, Xiong W, Baca-Regen L, Nagase H, Baxter BT. Mecha-
nism of inhibition of matrix metalloproteinase-2 expression by
doxycycline in human aortic smooth muscle cells. J Vasc Surg
2003;38:1376–83.
23 Villarreal FJ, Griffin M, Omens J, Dillmann W, Nguyen J, Covell
J. Early short-term treatment with doxycycline modulates postin-
farction left ventricular remodeling. Circulation 2003;108:1487–92.
24 Golub LM, Ramamurthy NS, McNamara TF, Greenwald RA,
Rifkin BR. Tetracyclines inhibit connective tissue breakdown:
new therapeutic implications for an old family of drugs. Crit Rev
Oral Biol Med 1991;2:297–321.
25 Chow AK, Cena J, Schulz R. Acute actions and novel targets of
matrix metalloproteinases in the heart and vasculature. Br J Phar-
macol 2007;152:189–205.
 2010 The Authors
Basic & Clinical Pharmacology & Toxicology  2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 108, 318–325
325
26 Rizzi E, Castro MM, Fernandes K, Barbosa F Jr, Arisi GM,
Garcia-Cairasco N et al. Evidence of early involvement of matrix
metalloproteinase-2 in lead-induced hypertension. Arch Toxicol
2009;83:439–49.
27 Dao HH, Lemay J, de Champlain J, deBlois D, Moreau P. Nor-
epinephrine-induced aortic hyperplasia and extracellular matrix
deposition are endothelin-dependent. J Hypertens 2001;19:1965–
73.
28 Souza-Tarla CD, Uzuelli JA, Machado AA, Gerlach RF, Tanus-
Santos JE. Methodological issues affecting the determination of
plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities.
Clin Biochem 2005;38:410–4.
29 Gerlach RF, Uzuelli JA, Souza-Tarla CD, Tanus-Santos JE.
Effect of anticoagulants on the determination of plasma matrix
metalloproteinase (MMP)-2 and MMP-9 activities. Anal Biochem
2005;344:147–9.
30 Gerlach RF, Demacq C, Jung K, Tanus-Santos JE. Rapid separa-
tion of serum does not avoid artificially higher matrix metallopro-
teinase (MMP)-9 levels in serum versus plasma. Clin Biochem
2007;40:119–23.
31 Castro MM, Rizzi E, Rodrigues GJ, Ceron CS, Bendhack LM,
Gerlach RF et al. Antioxidant treatment reduces matrix metallo-
proteinase-2-induced vascular changes in renovascular hyperten-
sion. Free Radic Biol Med 2009;46:1298–307.
32 Garcia RA, Go KV, Villarreal FJ. Effects of timed administration
of doxycycline or methylprednisolone on post-myocardial infarc-
tion inflammation and left ventricular remodeling in the rat heart.
Mol Cell Biochem 2007;300:159–69.
33 Griffin MO, Jinno M, Miles LA, Villarreal FJ. Reduction of myo-
cardial infarct size by doxycycline: a role for plasmin inhibition.
Mol Cell Biochem 2005;270:1–11.
34 Vinet L, Rouet-Benzineb P, Marniquet X, Pellegrin N, Mangin L,
Louedec L et al. Chronic doxycycline exposure accelerates left
ventricular hypertrophy and progression to heart failure in mice
after thoracic aorta constriction. Am J Physiol Heart Circ Physiol
2008;295:H352–60.
35 Ceron CS, Castro MM, Rizzi E, Montenegro MF, Fontana V,
Salgado MC et al. Spironolactone and hydrochlorothiazide exert
antioxidant effects and reduce vascular matrix metalloproteinase-2
activity and expression in a model of renovascular hypertension.
Br J Pharmacol 2010;160:77–87.
36 Prall AK, Longo GM, Mayhan WG, Waltke EA, Fleckten B,
Thompson RW et al. Doxycycline in patients with abdominal
aortic aneurysms and in mice: comparison of serum levels and
effect on aneurysm growth in mice. J Vasc Surg 2002;35:923–9.
37 Saivin S, Houin G. Clinical pharmacokinetics of doxycycline and
minocycline. Clin Pharmacokinet 1988;15:355–66.