Overexpression of miR-19b Impairs Cardiac Development in Zebrafish by Targeting ctnnb1

Abstract

Background:
MicroRNAs are broadly accepted as crucial regulators of cardiovascular development, and dysregulation of their expression has been linked to cardiac disease. MicroRNA cluster miR-17-92 has been implicated in cardiac development and function, yet its defined mechanisms of action in this context are uncertain. Here, we focused on miR-19b, a key component of the miR-17-92 cluster proven to induce cardiomyocyte proliferation in vitro. We aimed to identify the biological significance of miR-19b in cardiac development and its underlying molecular mechanism of action in vivo.

Methods:
We micro-injected zebrafish embryos with different concentrations (0, 2, 4 and 8 μm) of miR-19b mimics or a negative control, and assessed the embryo malformation rate, mortality rate, hatching rate and heart abnormalities at 72 hours post-fertilization (72 hpf).

Results:
We found that overexpression of miR-19b impacted left-right symmetry and cardiac development of zebrafish embryos, characterized by pericardial edema, slower heart rate and cardiac looping defects in a dose-dependent manner. Moreover, several important signaling molecules in the Wnt signaling pathway were abnormally expressed, suggesting that overexpression of miR-19b induces the inhibition of the Wnt signaling pathway by directly targeting ctnnb1. Interestingly, the deformed cardiac phenotype was partially rescued by treatment with the GSK3β inhibitor lithium chloride.

Conclusion:
Our findings suggest that miR-19b regulates laterality development and heart looping in zebrafish embryos by targeting ctnnb1.

Full text

Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1988
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
1421-9778/14/0336-1988$39.50/0
Original Paper
Copyright © 2014 S. Karger AG, Basel
Accepted: May 16, 2014
This is an Open Access article licensed under the terms of the Creative Commons Attribution-
NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to
the online version of the article only. Distribution permitted for non-commercial purposes only.
Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital of Nanjing
Medical University, No. 123 Tian Fei Xiang, Mo Chou Road, Nanjing 210004 (China)
E-Mail: shupinghan@njmu.edu.cn and E-Mail zhangbinyu@njmu.edu.cn
Tel. +86-025-52226561, Fax +86-025-52226561
SP Han, ZB Yu
Overexpression of miR-19b Impairs Cardiac
Development in Zebrafish by Targeting
ctnnb1
Mengmeng Li Xiaoshan Hu Jingai Zhu Chun Zhu Shasha Zhu Xuehua Liu
Jing Xu Shuping Han Zhangbin Yu
State key Laboratory of Reproductive Medicine, Department of Pediatrics, Nanjing Maternity and Child
Health Care Hospital Afliated to Nanjing Medical University, Nanjing, China
Key Words
miR-19b • Zebrash • Cardiac development • ctnnb1
Abstract
Background: MicroRNAs are broadly accepted as crucial regulators of cardiovascular
development, and dysregulation of their expression has been linked to cardiac disease.
MicroRNA cluster miR-17-92 has been implicated in cardiac development and function, yet
its dened mechanisms of action in this context are uncertain. Here, we focused on miR-19b,
a key component of the miR-17-92 cluster proven to induce cardiomyocyte proliferation in
vitro. We aimed to identify the biological signicance of miR-19b in cardiac development and
its underlying molecular mechanism of action in vivo. Methods: We micro-injected zebrash
embryos with different concentrations (0, 2, 4 and 8 μm) of miR-19b mimics or a negative
control, and assessed the embryo malformation rate, mortality rate, hatching rate and heart
abnormalities at 72 hours post-fertilization (72 hpf). Results: We found that overexpression
of miR-19b impacted left–right symmetry and cardiac development of zebrash embryos,
characterized by pericardial edema, slower heart rate and cardiac looping defects in a dose-
dependent manner. Moreover, several important signaling molecules in the Wnt signaling
pathway were abnormally expressed, suggesting that overexpression of miR-19b induces
the inhibition of the Wnt signaling pathway by directly targeting ctnnb1. Interestingly, the
deformed cardiac phenotype was partially rescued by treatment with the GSK3β inhibitor
lithium chloride. Conclusion: Our ndings suggest that miR-19b regulates laterality
development and heart looping in zebrash embryos by targeting ctnnb1.
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Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1989
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
Introduction
Congenital heart disease accounts for nearly one-third of all major congenital
anomalies, representing a considerable health problem [1]. Disturbances in cardiac left–
right (LR) patterning signaling pathway(s) are thought to be a major contributor toward the
development of these anomalies [2]. It is widely accepted that the formation of the mature
vertebrate heart involves the complex orchestration of gene expression. Numerous genes
are known to be critical for cardiac morphogenesis, although their exact functions and their
integration with other cardiac regulators are poorly understood [3]. A newly recognized
regulator of cardiovascular development and function are microRNAs (miRNAs) [4]. MiRNAs
are a class of ~22-nucleotide non-coding RNAs that regulate the expression of protein-coding
 
however, the biological functions of many of them remain unknown.
           

is a key component of the miR-17-92 cluster that induces cell proliferation and oncogenic
growth [8]. These studies suggest that miR-19b has a role in cardiovascular development
    
19b can induce developmental abnormalities and cardiac LR asymmetric defects has not
been explored in vivo.
Cardiac development is a complicated and elaborate biological process that goes

and differentiation [9]. This process involves a number of signaling pathways, such as the aryl
hydrocarbon receptor (AhR), Wnt and retinoic acid (RA) signaling pathways [10–12]. Recent
studies have revealed that Wnt signaling has a complex array of functions in cardiovascular
development and heart morphogenesis, expanding the role for these pathways beyond cell

stem/progenitor cell self-renewal and differentiation [13]. It has also been established that
          
and cardiac LR patterning [14]. Thus, we hypothesized that overexpression of miR-19b can
induce developmental abnormalities and cardiac LR asymmetric defects in vivo through the
Wnt signaling pathway.
             
assessed cardiac developmental phenotypes and changes in the Wnt signaling pathway,
in order to identify the potential molecular mechanism that contributes to the effect of

model because they are an attractive and widely used vertebrate model for studying
    
and has been shown to be morphologically and physiologically similar to that of mammals
[15, 16]. Additionally, severe defects in the heart do not lead to immediate lethality as in many
vertebrate models, and even in the total absence of blood circulation they receive enough
oxygen by passive diffusion to survive and continue to develop in a relatively normal fashion
for several days, thereby allowing a detailed analysis of animals with severe cardiovascular
            
the human heart in many respects, such as migration of cardiac precursor cells towards the
central line, heart tube formation, early chamber formation and the looping process [18, 19].

and their underlying molecular mechanisms.
Materials and Methods
Animal husbandry
     
Animal Research Center (MARC), Nanjing University in accordance with an Institutional Animal Care and
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Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1990
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
Use Committee (IACUC)-approved protocol. Embryos were obtained from the natural spawning of wild-type
(WT) adults, and were grown at 28°C ± 1°C in embryo medium as previously described [20]. Morphological
  
Embryos older than 24 h post-fertilization (hpf) were incubated in 0.003% phenylthiourea to inhibit
pigment formation.
MiRNA mimics and microinjection
              
              
  
          
needles. Following microinjection, embryos were incubated at 28°C ± 1°C, and solutions were changed and
degenerating embryos removed daily.

After microinjection, we determined the hatching rate at 72 hpf, and malformation and lethality rates
         

 

Measurement of heart rate

The stock tricaine solution (pH 7) was made as follows: 400 mg tricaine powder, 97.9 ml double-distilled

8 ml of clean tank water. The anesthetized embryos were then transferred to a recording chamber perfused
2PO4, 1.8 mmol/l CaCl2,
1 mmol/l MgCl2 

This was conducted under a dissecting microscope in 20-s intervals.

RNA probes for in situ hybridization were generated for the following genes: atrial myosin heavy
chain (amhc), ventricular myosin heavy chain (vmhc), cardiac myosin light chain-2 (cmlc2), natriuretic
peptide precursor A (nppa), notch homolog 1b (notch1b), bone morphogenetic protein 4 (bmp4), catenin
 

      



probe synthesis are listed in Table 1.
Whole-mount in situ hybridization

               
paraformaldehyde at 4°C for 12–16 h, before serial dehydration through graded methanol solutions (25%,
50%, 75% and 100%) and storage at -20°C for at least 30 min. Hybridization was performed overnight at


 
and experimental embryos were processed in parallel. In situ images were captured using an Olympus DP71
digital camera (Olympus, Tokyo, Japan).
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75.101.163.131 - 8/25/2014 10:00:58 PM

Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1991
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry

 


     
    

 
       




 described [25, 26]. Total RNAs were reversely transcribed
using the miR-19b 
 U6, forward
       
 
Luciferase assay
To generate luciferase miR-19b target reporters, Oligonucleotides corresponding to a portion of the

  
      
   
              
   
 
           
     
normalized to Renilla luciferase.
Lithium chloride treatment
Lithium chloride (LiCl) treatment was carried out as previously described [27].
Table 1. Description of probes used for in situ hybridization
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Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1992
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
Statistical analysis
      2        
              

Results


evaluated the effect of increasing concentrations of miR-19b mimic on mortality and hatching
rates at different development stages (24, 36, 48, 72 and 96 hpf). Our results show that the
      
mimic–injected embryos, compared with wild-type (WT) embryos and NC-injected embryos
(Table 2). The hatching rates in miR-19b mimic–injected embryos decreased when compared
with the rates in the WT and NC groups (Table 3). This data shows that with increasing miR-
   
         
Table 2. -

Fig. 1. Overexpression of miR-19b in-
     
in a dose-dependent manner. The effect

mortality is shown. The mortality rates
ranged from 19% to 100% in embryos
injected with miR-19b mimics. The data
shows that with an increasing concentra-
tion of miR-19b mimic, the mortality rate
    

Table 3.  

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75.101.163.131 - 8/25/2014 10:00:58 PM

Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1993
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry



To select a suitable concentration for future work, the effects of increasing concentrations
of miR-19b mimic on malformation rates at different stages (24, 36, 48, 72 and 96 hpf)
             
with gross morphological deformities, including small size and pericardial edema (Fig. 2).
   
    
the malformation rates ranged from 40.47% to 100% in miR-19b mimic–injected embryos
compared with WT and NC-injected embryos (Table 4). After injection with 4 µm miR-19b
mimic, the phenotypes were visible. No embryos survived to 72 and 96 hpf after an injection
of 8 µm miR-19b mimic.

             
  
Fig. 2. -


of 3.2 times. Red arrows: pericardial edema.
Fig. 3. Overexpression of miR-19b
increased the malformation rates of
-
ner. The effect of miR-19b mimic in-
   
is shown. The rates of malformation
ranged from 40.74% to 100% in em-
bryos injected with miR-19b mimics.
The data shows that with increasing
concentrations of miR-19b mimic,
 
   
dose-dependent effects.
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Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1994
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
to investigate its effect on cardiac development. In normal cardiac development, the linear
heart tube has formed at 24 hpf, and the tube lies along the anterioposterior axis, with

chambers have formed by 30 hpf, and the heart undergoes looping morphogenesis by 36
hpf, with functional valves formed by 48 hpf [17]. At 48 hpf, the early cardiac development
Table 4. 

Fig. 4. Expression of miR-19b is
up-regulated in miR-19b mimic in-

validation of miR-19b expression
in WT, miR-19b mimic injected and

and 48 hpf. Data are representative
 
data are expressed as the means ±
     

Fig. 5. -

 
 -
 
 

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75.101.163.131 - 8/25/2014 10:00:58 PM

Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1995
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry

96 hpf to assess cardiac development. As shown in Fig. 5, obvious abnormalities in heart
morphology were observed in miR-19b–injected embryos, including severe pericardial
            
that we observed in the WT and NC-injected embryos, the heart chambers from miR-19b–
injected embryos were string-like and elongated. In the WT or NC group, the ventricle and
atrium overlapped and were not distinguishable in the lateral view. In contrast, ventricles of
the treated embryos were positioned anterior to the atrium, so the chambers could be easily
distinguished with little overlap. These results indicated that miR-19b caused abnormal



heart rate was examined at 48, 72 and 96 hpf to evaluate the effects of overexpression of miR-
19b on cardiac contraction. As shown in Fig. 6, the mean heart rates at 36 hpf were 145.1 ±
2.6 beats per minute (bpm), 148.5 ± 4.9 bpm and 78.5 ± 3.9 bpm for WT, NC-injected and 4

were 158.1 ± 4.6 bpm, 158.5 ± 4.9 bpm and 88.5 ± 3.9 bpm for WT, NC-injected and 4 µm

204 ± 6.4 bpm, 200.8 ± 6.2 bpm and 170 ± 9.3 bpm for WT, NC-injected and 4 µm miR-19b

± 3.7 bpm, 206.7 ± 3.5 bpm and 177.8 ± 3.4 bpm for the WT, NC-injected and 4 µm miR-19b


control embryos exhibited vigorous, rhythmic contractions, ensuring circulation throughout
the body. In the miR-19b mimic–injected embryos, irregular and weak contractions were
Fig. 6. -
jection with miR-19b on heart rate is shown. The heart rate was determined in embryos and larvae at 36 (A),

in miR-19b mimic–injected embryos when compared with controls.
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75.101.163.131 - 8/25/2014 10:00:58 PM

Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1996
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
observed readily. Taken together, these data indicate that overexpression of miR-19b leads
to a remarkable change in the contractile function of the heart.

To further investigate the effect of miR-19b overexpression on cardiac chamber and
atrioventricular canal development at the molecular level, the expressions of amhc, vmhc
and cmlc2 were examined at 48 hpf by whole-mount in situ hybridization. Normally, amhc is
expressed in the atrium, vmhc is expessed in the ventricle, and cmlc2 is expressed throughout
the heart. Compared to control embryos, embryos injected with miR-19b mimic showed an
abnormal expression pattern of vmhc, amhc and cmlc2, with LR defects, including reversed


however, the expression of atrioventricular canal marker genes was not changed in embryos
injected with miR-19b (Fig. 8). Taken together, this data demonstrates that overexpression

on heart valve development.

In order to better understand the mechanism underlying overexpression of mir-
19b on cardiac development, we studied the Wnt signaling pathway, which is divided
Fig. 7. Overexpression of miR-19b alters the expression of cardiac chamber marker genes. Representative

-
ward movement of the heart (jogging) is followed by rightward bending of the ventricle (D-looping) in WT
 



blue arrow: atrium.
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Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1997
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
           
catenin pathway) is the pathway in which Wnt binds to and activates the frizzled seven-
transmembranespan receptor. Next, axin is removed from the “destruction complex”, and

transcription [28, 29]. Recent studies have suggested that Wnt signaling plays a critical role in
the embryonic development of a variety of organisms, and that the functions of Wnt signaling

 

of lft2, foxj1a and gata4, and found them to be abnormally expressed in embryos injected
             
mir-19b mimic–injected embryos, while foxj1a expression in dorsal forerunner cells (DFCs)
was markedly downregulated and gata4 expression was expanded caudally and laterally.
These results indicate that overexpression of mir-19b alters the Wnt signaling pathway in

MiR-19b directly targets ctnnb1


Fig. 8. Overexpression of miR-19b has no effect on the expression of atrioventricular canal marker genes.



and scope, except for changes in expression position due to LR defects when compared with WT embryos (A,

embryos examined with the representative expression/total number of embryos are presented. Red arrow:
atrioventricular canal.
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Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1998
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
in DFCs, interrupts normal asymmetry of the heart [31]. To further investigate this potential

construct (Fig. 10C). We found that luciferase activity was decreased after microinjection
          
  

            

the inhibition of the Wnt signaling pathway by directly targeting ctnnb1.

activation of the 
As shown in the data mentioned above, we hypothesized that inhibition of Wnt signaling
     
development, especially cardiac development. In order to test this hypothesis, we examined
whether activating the Wnt signaling pathway could rescue the miR-19b mimic–injected

            
induced cardiac anomalies morphologically and by in situ hybridization (Fig. 11). We found
that treatment with LiCl partially rescued the cardiac defects caused by overexpression of
mir-19b, including pericardial edema, heart looping defect and cardiac asymmetry. We also
Fig. 9. Overexpressi-
on of miR-19b altered
lft2, foxj1a and gata4
expression. Represen-
tative images of lft2 ex-
pression in the cardiac

dorsal view of 22-so-
mite–stage embryos,
with the anterior end
at the top. The expres-
sion position of lft2
was altered in miR-19b
mimic–injected em-
bryos (C), compared
with WT (A) and NC-
  
Representative images
of foxj1a expression in
DFCs are also shown.
Foxj1a expression in
DFCs was severely
downregulated in miR-
19b–overexpressing

compared with WT (D)
 


miR-19b (I). The asterisk indicates alteration of gata4 expression.
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Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 1999
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
Fig. 10. MiR-19b directly targets ctnnb1. A representative image of ctnb1 expression in 48 hpf embryos
determined by in situ hybridization is shown (A). The expression in the miR-19b mimic–injected embryos
      


assay 

   
partially rescued by LiCl through appropriate activation of the Wnt signaling pathway.
Discussion
   
and tumor growth, with individual members of the cluster appearing to possess distinct
functions [33–35]. For example, inhibition of miR-17 after hypoxia-induced hypertension
rescued right ventricle hypertrophy [36], whereas miR-18 and miR-19 are downregulated

regulation of the miR-17-92 cluster expression levels to maintain normal heart development
and tissue homeostasis. In this study, we focused on miR-19b, which is a key component of
the miR-17-92 cluster proven to induce cardiomyocyte proliferation in vitro [7].

    
  
             
development. Our results show that overexpression of miR-19b increased embryonic lethality

levels of miR-19b during the early developmental period exhibited gross malformations, such
as small size and pericardial edema. Additionally, an abnormal embryonic heart phenotype

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Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 2000
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
Fig. 11. Treatment with LiCl partly rescues
cardiac defects caused by overexpression of
miR-19b. Representative images of cardiac
        
        
      
19b mimic–injected embryos displayed
     
defects (H), which were rescued by treat-
  
miR-19b injection (I and J). In contrast, car-
diac morphology and looping were normal

      
     
yellow arrow: pericardial edema.
exhibited abnormal positioning of the ventricle and atrium, defects in cardiac looping and
LR asymmetry. In addition to the structural defects described above, a decreased heart rate
was observed in miR-19b mimic–injected embryos. However, the trajectory of heart rate was
increased in miR-19b mimic–injected embryos. This indicated that overexpression of miR-
19b impaired the function of the heart, which exhibited developmental delay. In summary,

of miR-19b impacts normal cardiac development, resulting in congenital heart disease.
           
embryonic heart development, we also wanted to study its molecular mechanism. It is
known that multiple signaling pathways converge to regulate heart development, such as

shown to play an important role in cardiac development. The Wnt proteins are a group of
 
pathways in the cell that can be categorized into canonical and non-canonical Wnt pathways
           
     

expression of several key molecules in the canonical Wnt signaling pathway after miR-19b
mimic injection. In agreement with our hypothesis, our results indicated that inhibition of
the canonical Wnt signaling pathway contributes to cardiac developmental abnormalities in
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75.101.163.131 - 8/25/2014 10:00:58 PM

Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 2001
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry

We next found that ctnnb1 is a functional target of the miR-17-92 cluster. The ctnnb1

to be important in embryonic development. For example, knockdown of ctnnb1 in the whole

link the function of miR-19b and ctnnb1 in LR asymmetric heart development. Future
studies will be important to illustrate how the miR-19b–ctnnb1 axis participates in cardiac
LR asymmetry.
In summary, these studies demonstrate that overexpression of miR-19b is detrimental

We were able to delineate the molecular mechanisms underlying this effect, leading to the

through inhibition of the canonical Wnt signaling pathway.
Acknowledgments
   
           



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
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
3 
4 
5 
6 

7 
8 


9 

10 

11 

12 
13 

14 
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15 
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16 
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75.101.163.131 - 8/25/2014 10:00:58 PM

Cell Physiol Biochem 2014;33:1988-2002
DOI: 10.1159/000362975
Published online: July 01, 2014
© 2014 S. Karger AG, Basel
www.karger.com/cpb 2002
Li et al.: Overexpression of miR-19b Impairs Cardiac Development
Cellular Physiology
and Biochemistry
Cellular Physiology
and Biochemistry
17 

18 
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19 

20 

21 
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22 
   
23 

24 
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25 
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26 
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27 

28 

29 

30 

31 

32 

33 


34 


35 
36 


37 

improves lung and heart function in experimental pulmonary hypertension. Am J Respir Crit Care Med
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38 


39 

40 

41 Puga A: Perspectives on the potential involvement of the AH receptor-dioxin axis in cardiovascular disease.

42 
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