226
Journal of Huazhong Univer.sityof Science and Technology[Med Sci] 9 9 ~# ~ X ~ ~- ~ [ ~ ~ ( ~ i~, ~ ) ~i ]
24 (3): 226-228, 2004
Effect of Retinoic acid on Platelet-derived Growth Factorand Lung Development in Newborn Rats CHEN Hongbing ( ~ ~ ~ ) , CHANG Liwen ( "g"~ ~ ) , LIU Hanchu ( #] ~s ~ ), RONG Zhihui ( ~ , ~ ~ ), ZHU Huaping (g~t,tg-~), ZHANG Qianshen (~i,t~'~), LI Wenbin (4a :~a~.) Department o f Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
Summary: The influence of platelet-derived growth factor (PDGF) on lung development in newborn rats and the effect of retinoic acid (RA) on PDGF in lung development were investigated. Newborn Sprague-Dawley (SD) rats were randomly assigned to two groups: control group and RA group. The rats in RA group was intraperitoneally injected with all trans-retinoic acid (500 ~tg/kg every day) for consecutive 3 days after birth, while those in the control group were not subjected to intervention. Immunohistochemical assay was performed to locate the expression of PDGF. mRNA levels of PDGF were measured by reverse transcription polymerase chain reaction (RT-PCR) a t age of 1, 3, 5, 7, 10, 14, 21 days. The method of radial alveolar counts (RAC) was used to measure the amount of the alveoli of the lungs. It was found that with increasing days, levels of PDGF-A and PDGF-B changed to verying degrees. RA could elevate significantly the expression levels of PDGFA mRNA and protein (P%0.01), but not affect the expression levels of PDGF-B mRNA and protein markedly (P~>0.05). It is suggested that PDGF might play an important role in lung development. RA can stimulate lung development through increasing the expression levels of PDGF-A mRNA and protein. Key words: platelet-derived growth factor; retinoic aicd; lung development; newborn rats Both Retinoic acid ( R A ) and platelet derived g r o w t h factor ( P D G F ) play an important role in lung d e v e l o p m e n t , hut relation between t h e m and mechanism by which they influence lung development remained unclear. T h e purpose of this study was to investigate the effect of R A and P D G F on postnatal lung tissue and cells through animal experimentation in order to illustrate their interrelation and the possible mechanism intervening postnatal lung development at level of molecule and gene.
1
MATERIALS AND METHODS
1.1 Establishment of Lung Development Model and Preparation of Lung Specimens in Newborn Rats Spragne-Dawley ( S D ) rats aged 2 days were assigned randomly to two groups: control group and RA group. T h e rats in R A group was intraperitoneally injected with R A (500 / , g / k g every day) for consecutive 3 days after birth, while those in control group were not subjected to intervention. At postnatal 1, 3, 5, 7, 10, 14, 21 day, 8 newborn rats in each group were anesthetized, the lung was perfused in situ via a tracheal cannula with 4 buffered formalin and fixed for 15 min, then lung was excised and immersed in formalin for up to 24 h, then embedded in paraffin for further study. Frozen lung samples were preserved for 24 h in liquid nitrogen, then in - - 7 0 ~ refrigeratory. 1.2 Methods 1.2. 1 Radial Alveolar Counts (RAC) According tO the method described by Jakkula E~ , radial alveolar counting was performed by determining the number of septae that intersected a perCHEN Hongbing, male, born in 1963, Postgraduate
pendicular line drawn from the center of a respiratory bronchiole to the distal acinus (connective tissue septum or pleura). At least 5 counts were performed on each lung section. 1 . 2 . 2 Immunohistochemical Detection of PDGF T h e paraffin-embedded lung sections (5 ~ m ) from all groups were immunostained according to standard procedures by using polyclonal antibody of P D G F - A , -B ( W u h a n Boster C o m p a n y , China) and SP Kits (Beijing Zhongshan C o m p a n y , China). M H I A S 2000 was used to measure values of average luminosity, which can semiquantitate and reflect the intensity of expression of PDGF. 1 . 2 . 3 Extraction of Total RNA and RT-PCR T h e sequences of the primers were as follows c8 ( W u h a n Saibaisheng c o m p a n y , China) : F o r w a r d primer of P D G F - A : 5'-CTC G G C T G C G G A T A C C T C GC-3"; Reverse primer: 5"-CTT G A G G G C T G G CAC T T G ACG C-3", with an expected size of the amplified sequence of 403 bp. Forward primer of PDGF-B: 5"GCT C C T T T G A T G ACC T T C AGC-3"; Reverse primer: 5"-CAG CCC G A G CAG CGC T G C ACC TC-3", with an expected size of the amplified sequence of 282 bp. F o r w a r d primer of ~-actin: 5"-ATC T G G CAC CAC ACC T T C T A C A A T GGC T G C G-3'; Reverse primer: 5 ' - C G T C A T A C T CCT G C T T G C T G A T C C ACA T C T GC-3", with an expected size of the amplified sequence of 838 bp. Total R N A was extracted from 50 mg tissue using 1 ml Triozl reagent by following the manufacturer's instruction. Reverse transcription ( R T ) reaction was performed in 25/~1 preparation. It was incubated at 37 ~ for 1 h, and at 95 ~ for 5 min to inactivate the reverse-transeripase. T h e resultant R T mixture was eDNA. PCR amplification was carried out at 50 gl re-
CHEN Hongbing el a/. tinoic acid on Platelet derived G-r{}wth Factor and I.ung I)cvelopment
w e r e i n c r e a s e d m a r k e d l y . I.ung m o r p h o l o g y was c h a r a c t e r i z e d t)y s m a l l e r air cavity and t h i n n e r w a l l , and m e s e n c h y m a reduced or d i s a p p e a r e d . R A g r o u p s : ( ' o m p a r e d wit}] c o n t r o l g r o u p , the n u m b e r of a l v e o l a r w e r e i n c r e a s e d u n i v e r s a l l y at each time point.
action solution. ] ' h e t u b e s were phlced in the I ) N A t h e r m a l cycler and p e r f o r m e d , followed by 30 cycles of f o l l o w i n g s e q u e n t i a l s t e p s : d e n a t u r a t i o n for 3 rain at 94 C , a n n e a l i n g for I rain at {;0 ( 9 and e x t e n s i o n for 1 rain al 72 (_ . F i n a l i n c u h a t i { } n Ias ted 10 rain at 72 ( (final e x t e n s i o n ) . A l i q u o t s of t h e I } C R p r o d u c t s were a n a l y z e d by electrophoresis
on 1.5
2.2
2.3 D y n a m i c C h a n g e in the E x p r e s s i o n o f P D G F in Lung Tissue T h e a v e r a g e l u m i n o s i t i e s of P I ) G F - A w e r e all s i g n i f i c a n t l y h i g h e r in R A g r o u p s t h a n in c o n t r o l g r o u p s at day 1. ,+3, 5, 7, 10, 14, 21 ( P ~ 0 . 0 1 ) . Bu~ t h e r e was n() s i g n i f i c a n t d i f f e r e n c e in a v e r a g e l u m i n o s i t i e s of t ) I ) G F - B at each t i m e point b e f o r e and a f t e r R A t r e a t m e n t ( P ~ 0 . 0 5 , t a b l e 2).
RESULTS
2.4 2. 1
Lung Morphology
RAC of newborn rats at day 7, 10. 14, 21 ( x-Fs, n = 8 )
RAt"
Groups l)ay 7 Control
1. 12 f 0 .
I2
RA 5.73 t-(}.51 9 P~'0.01 as COml}arv{I with control groups Table 2
ltem.s
Day ]
PDGF-B
Control
(}. 2293
r 0. ()1138
RA
0.58 ~1 } 0. {>587
Control
O. 1{;27
RA
Day 1{}
Day II
Day 21
7 . 0 1 I 0. 17
8.65 ~ 0.96
I(5.2~ 1.73
8.88 ; 0 . 5 3
10.75 ] - 0 . 8 2
17.8+1.8{.)'
Average luminosity of PD{;F-A and PDGF-B at various time points ( x + s , n = 8 )
Groups
PDGF-A
Electrophoresis of PDGF-A mRNA
P I X ; F A m R N A was markedly higher in R A groups than in control groups at day 1, 3, 5, 7, 10, 14 ( P < (). 0 1 ) , but lhere was no difference at day 21 between them ( P > 0 . 05). H o w e v e r , there was no significant difference in P I X ; F - B m R N A between R A g r o u p and c o n t r o l grout) ( P : : > 0 . 0 5 , t a b l e 3 ) .
C o n t r o l g r o u p s : At postrmtal 1 d a y , lung s t r u c t u r e p r e s e n t e d l c r m i n a I sac, l a r g e r air c a v i t y and t h i c k e r wall and fewer a l v e o l u s : A f t e r 7 d a y s , the n u m b e r of a l v e o l u s were hwrcasc(I; ;it I,lth and 21st d a y the a l v e o l i , the basic s t r u c t u r e of l u n g . 1"able I
Radial Alveolar Counts (RAC)
At day 7, 1{), 1'1, 21, c o m p a r e d w i t h c o n t r o l g r o u p . RA{" was s i g n i f i c a n t l y increased in R A g r o u p ( P < ~ 0 . 0 1 , t a b l e 1).
% a g a r o s e gel a n d v i s u
alized w i t h an uhravi{}let transillumitmt{)r by ethid ium b r o m i d e staining. T h e r e s u h s were a n a l y z e d by GDS-8000 ( U V P (%rot}any. ( ; N ) and t)hot(} graphed. 1.2. 4 Statistical Analysis The resuhs were e x p r e s s e d as . f • and the difference a m o n g g r o u p s w a s a s s e s s e d by a n a l y s i s of v a r i a m ' c or t test by using S P S S 10.0 package. 2
227
t 0. {1:~71
O. 2o78
F o. O273
Day :i {). 1529 I u.():~153 (). 7o32 {L 17tl (). 18ll + o {}:t!} I
(>. 1!}{;:t (). 0350
Av{'rage luminosity of PD(;F I)ay 7; l)av 7 l)ay 10
l)ay 14
{7. 359!)
0. 3264
(}. 057o (). 7343
1.0. (){~:i2 0. {;876
~_o. 08!}9 0. (~00l
§
[:0. o525 0. o515
-_ o. o638 {). 0932
~ o . o615' 0. 1758
+ o . 37(H" 0.0575
; u. 1 0 1 7 o. 06,:11 ~(). o336
O. 261!}
4-0.{}135 : 0 . 0 2 6 3
§
0. o{;13 i o. ()336
§
o. 1053 too. 0272
Day 21
0. 3711
0. 3119
0312 o. 7081
:,~:().0799 O. 3994 t- 0.0787' (}. 09!}3
+0.0252 o. 1815 o409
§
o. 109 0440
" P < 0 . 0 1 as compared wilh control Rroup Table 3
Items PI)GF-A
Changes of content of PDGF-A mRNA and PI}GF-B mRNA at various time points ( x + s . n = 8 )
Groups Control RA
l)ay i{
[)ay 5
0.3135
{L 39{~!}
0. 274!)
0. 3158
control RA
{). 3378 I62:1
•
Day 21 0. 9572
1242
q 0.0255
+(>. 1911
- 0 . o315
~ +7. 1032
0. t;q5,{)
{). 7]t)]
0. 871 I
o. 7()18
{). 8013
0. 8355
20. 1 9 2 8
~ 0. 1{77
+_0.065'
-~0.2066
:~().122
0.77 i{;
..L 0.0307 .
~(}. 13:-~!}
•
I)ay ].1, 0. 6568
' O. 1586 fi0.018!) PDGF-B
Average luminosity of PI)C;-F I)av 7 I)ay J.0
Day I
0. :,!):~8
(). :;3{){i
0. 1361
{).oSIU
-' {).()S] I
r ().PIGS
:t o. 181I
_-!-o. ]71!)
0.27,96 L0. 0633
(}" {~ I!~:', b0. I317
u. :-; 176 -1 o. O865
0. 121!} h0. 1129
(7.57:~8 t {7. 2833
{). 7527 ~{}. 009,1
0.2905 ~ 0 . 1058
(}. 5{';{;:',
0.95l 1 1. 0329 (}. 957l + 0 . 0582
" P ~ 0 . 0 1 as cornparcd whh comrol Kmu I) 3
DISCUSSION
It had been t e s t i f i e d that R A ('(add a c c e l e r a t e lung d e v e l o p m e n t ::~', but the m e c h a n i s m ren+mmed
unclear. R A can c h a n g e b i o a c t i v i t y of v a r i o u s s u b s t a n c e s in vivo by m o d i f y i n g and b e a r r e l a t i o n to lung m o r p h o l o g y d e v e l o p m e n t . P D ( ; F (:an s t i m u late lung b r a n c h i n g f o r m a t i o n . T h e r e f o r e , we p r e s u m e d that R A m i g h t have r e l a t i o n s w i t h P I ) G F .
228
Journal of Huazhong University of Science and Technology [Med Sci] 24 (3):226-228, 2004
In this study, term newborn rats were injected intraperitoneally with all trans-retinoic acid ( A T RA, 500 t2g/kg every day) at once after birth. PDGF-A m R N A was obviously increased after 24 h. After continuous intraperitoneal injection for consecutive 3 days, PDGF-A mRNA was significantly increased at 3 , 5 , 7 , 14 days, especially at day 5 and 14, PDGF-A m R N A reached its peak. T h e expression of PDGF-A protein was enhanced within two weeks too, and two peaks occurred at day 5 and 14 respectively. On the contrary, there was no significant difference in the expression of PDGF-B m R N A and protein between RA group and control group. Histomorphology revealed acinus buds and typical small-diameter alveolus were increased, and mesenchyma thinned in RA groups as compared with control groups. RAC showed the alveolar number at day 7, 10, 14, 21 was distinctly increased in RA groups as compared with control groups, suggesting that RA may stimulate cell proliferation by upregulating the expression of PDGF-A m R N A and protein either indirectly or directly. RA can enhance ongoing alveolarization by increasing the total number of alveoli formed E4?. RA, one of an important active metabolite of Vitamin A, can regulate the open and close of the correlative genes of lung morphology, and also upregulate the expression of growth factor ligand a n d / o r its receptor through its acting on RA-responsive genes encoding growth factors, which play a regulatory effect on lung development E4's~. It was reported that RA may promote RA-responsive DNA encoding, then RAR ( R A receptor) may be combined with RA response elements ( R A R E ) and further influence transcriptive frequency. R A R E locates in the promoter regions of PDGF-A and P D G F R , suggesting that RA upregulates the expression of PDGF-A m R N A at the transcriptional level C67" PDGF is a potent mitogenic agent and its four subunits have been identified to date, named P D G F - A , -B, -C, -D [7]. Both PDGF-A and PDGFB play an important role in lung development. Ligand exerts their action via binding to and dimerization of two types of receptor tyrosine kinases, designated a-receptors ( P D G F - R a ) and ~-reeeptors ( P D G F - R ~ ) . PDGF-A only combines with PDGFR-~, but PDGF-B can bind P D G F R - a , -13. Whether or not a given PDGF ligand-receptor interaction can occur in vivo depends critically on the spatial and temporal expression patterns of receptors and ligands, through signal PDGF dimers initiate signaling by inducing receptor dimerization and autophosphorylation. At present target cell research revealed that P D G F - A might play an important in alveolarization Esl. PDGF-A is a potent growth factor for mesenchymal cells in the developing lung. The lack of P D G F - A results in the absence of alveolar smooth muscle cells ( S M C ) , exhibiting reduced deposition of elastin fibres in the
lung parenehyma, and developing lung emphysema due to complete failure of alveogenesis, lessened lung volume and decreased terminal buds c<. Whereas, PDGF-A overexpression results in increased lung mesenchyme and lung size, failure of airspace development :< . These researches demonstrate that PDGF-A is an important factor regulating the growth of the lung and the development of the airspaces. Lung development is in crucial period within postnatal two weeks. During this stage, lung increases rapidly and perfects gradually in order to exert its breathing function. Full-formation of lung branches and septation and increases of lung mass, which is sign of lung maturation. PDGF-A and PDGF-B have close relation to lung maturation. PDGF-A is an indispensable factor in lung branching morphogenesis, while PDGF-B stimulates parenchymal cell multiplication. Both of two factors locate in parenehyma and mesenchyma. In this study, after intraperitoneal injection of RA in rats, it was observed that RA could upregulate appropriately the expression of PDGF-A m R N A and protein, but the expression of PDGF-B m R N A and protein didn't change markedly. F u r t h e r m o r e , the number of small-diameter and thin-wall alveolus were increased, indicating that RA promotes lung branches and alveolar formation through PDGF-A. PDGF-B is involved in the increase of lung mass, but there is uneonspicuous relation to morphology. REFERENCES
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