| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1Pediatrics and 5Molecular Pharmacology, Northwestern University, Chicago, Illinois 60611-3008; 2Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2650; and 3Department of Pediatrics and 4Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94143-0106
Submitted 15 November 2002 ; accepted in final form 24 March 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
|---|
vascular graft placement; shunted lambs
Recent evidence has demonstrated alterations in VEGF expression in a variety of pulmonary hypertensive disorders. For example, increased VEGF expression has been reported in newborns with congenital diaphragmatic hernia and pulmonary hypertension (34), and increased VEGF protein has been reported in the tracheal aspirates and type II pneumocytes of neonates with persistent pulmonary hypertension (PPHN) (23). Similarly, increased VEGF expression is reported in the lungs of adults with advanced pulmonary vascular disease secondary to congenital heart disease, with more pronounced expression in areas of advanced plexiform lesions (13, 17, 39). These observations correlate VEGF dysregulation with altered endothelial phenotype and abnormal vascularization. In addition, various clinical and in vivo studies on VEGF expression in models of increased blood flow have been reported. For instance, increased blood flow induced by bradycardia, or in response to exercise, induces a temporary increase in VEGF mRNA expression and protein secretion (24, 40). In vitro studies also support the concept that increased biomechanical forces, such as cyclic stretch and laminar shear stress, lead to increased VEGF expression (12, 15, 31, 37).
We have developed an animal model of pulmonary hypertension by inserting an aortopulmonary vascular graft in the late-gestational fetal lamb (3, 4, 28). Postnatally, these lambs have increased pulmonary blood flow and pressure. In addition, they display pulmonary vascular remodeling characterized by increased medial wall thickness of the small muscular pulmonary arteries and abnormal extension of muscle to peripheral pulmonary arteries (18, 27, 28). Last, at 4 wk of age, these lambs have a transient increase in the number of barium-filled intra-acinar pulmonary arteries, which may represent an early adaptive angiogenesis and/or vessel recruitment (28). We hypothesized that increased VEGF expression participates in pulmonary vascular remodeling secondary to increased pulmonary blood flow. Therefore, in the present study, we investigated the developmental changes in expression of VEGF and its two main receptors: Flt-1 and KDR/Flk-1 in 1-day-old and 1-, 4-, and 8-wk-old lambs with increased pulmonary blood flow.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
1
mg·kg-1·min-1) and diazepam (0.002 mg
· kg-1 · h-1), intubated with a 5.5-mm
outer diameter endotracheal tube, and mechanically ventilated with a
pediatric, time-cycled, pressure-limited ventilator (Healthdyne, Marietta,
GA). Ventilation with 21% O2 was adjusted to maintain an arterial
PCO2 (PaCO2) between 35
and 45 Torr. Via a midsternotomy incision, the lambs were instrumented to
measure pulmonary and systemic arterial pressure, right and left atrial
pressure, heart rate, left pulmonary blood flow, and oxygen saturations as
previously described (28).
Then, at 1 day and at 1, 4, or 8 wk after birth, the lambs were euthanized
with an intravenous injection of pentobarbital sodium (Euthanasia CII; Central
City Medical, Union City, CA) and subjected to bilateral thoracotomy. An
autopsy was performed to confirm patency of the vascular graft. The lungs were
removed and prepared for RNA preparation, Western blot analysis, and
immunohistochemistry. All procedures and protocols were approved by the
Committee on Animal Research of the University of California, San Francisco
and Northwestern University. Tissue preparation for immunohistochemistry. The heart and lungs were removed en bloc. The lungs were dissected with care to preserve the integrity of the vascular endothelium. Sections (2–3 g) from each lobe of the lung were removed. These tissues were snap-frozen in liquid N2 and stored at -70°C until analysis.
For immunohistochemistry, the pulmonary vascular tree was rinsed with cold (4°C) PBS to remove blood and was fixed by perfusion with cold (4°C) 4% paraformaldehyde. The pulmonary artery was then clamped. The airways were fixed at 20 cm of H2O pressure by filling the trachea with cold (4°C) 4% paraformaldehyde. When the lungs were distended at this pressure, the trachea was clamped. The lungs were fixed for 24 h at 4°C by immersion in 4% paraformaldehyde. Representative slices from each lobe were removed, placed in 30% sucrose until they sank, placed in optimum cutting temperature compound, frozen on dry ice, and stored at -70°C until they were sectioned. Five- to ten-micrometer sections were cut using a cryostat, transferred to aminoalkylsilane-treated slides (Superfrost Plus; Fisher Scientific, Santa Clara, CA), and stored at -70°C (5).
Generation of an ovine VEGF antiserum. The ovine VEGF (oVEGF) protein sequence differs greatly from human VEGF. Therefore, an oVEGF-specific antiserum was prepared by injecting rabbits with a highly antigenic protein fragment corresponding to oVEGF120 (sequence: NH2-GCRIKPHQSQHIGEMSFLQHNK-COOH). Rabbits were bled at 6, 8, and 10 wk, and the 8-wk bled was immunopurified (BioSynthesis, Louisville, TX). The specificity of the antiserum was assessed by Western blot analysis using positive control generated from COS-7 cells transfected with an oVEGF-pCDNA3 construct. The cell media provided a secreted protein around 25 kDa. The rabbit anti-oVEGF sera did not cross-react with human VEGF.
Western blot analysis. Immunoblotting was performed as previously
described (3,
4). Protein extracts (100
µg) were separated on 4–20% (VEGF) and 7.5% (for VEGF receptors Flt-1
and KDR/Flk-1) SDS-polyacrylamide gel and electrophoretically transferred to
polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). The
membranes were blocked with 5% nonfat dry milk in Tris-buffered saline
containing 0.1% Tween 20 (TBS-T). After 1 h of blocking, the membranes were
incubated at 4°C for two consecutive days with 1:500 (3.4 µg/ml)
dilution of rabbit polyclonal anti-oVEGF in blocking solution. Alternatively,
membranes were probed with goat polyclonal antibodies against rabbit
polyclonal antibody against mouse Flt-1 (2 µg/ml, Santa Cruz Laboratories)
and goat polyclonal antibody against human KDR/Flk-1 (1 µg/ml, R&D
Systems) for 2 h at room temperature. Membranes were washed 3 x 15 min
with TBS-T and then hybridized with anti-rabbit horseradish peroxidase
antibody for 45 min. After 3 x 15 min washes, bands were visualized with
chemiluminescence using a Kodak Digital Science Image Station (NEN, Boston,
MA). The following size bands were obtained: 25 kDa for monomeric VEGF,
200–220 kDa for KDR/Flk-1, and 130 and 180 kDa for Flt-1. For Flt-1, the
130-kDa band was found to be nonspecific.
To compare the various protein levels obtained from controls and shunts
(n = 5, total of 10 samples per age) of various ages (1-day-old and
1-, 4-, and 8-wk-old, total of 40 samples), four different gels were run (1
for each age containing protein samples from both control and shunted lambs).
Also, included on each gel was an internal control (a lung extract prepared
from a 1-day-old shunted lamb). Each of the four membranes was probed for the
particular protein of interest and then reprobed the next day for
-actin. Each densitometric value was divided by its -actin control
to obtain a relative value for VEGF, Flt-1, and Flk-1 protein levels. Relative
values were averaged and then corrected using a factor (relative protein level
of internal control obtained from that particular blot divided by relative
protein level of internal control from the original day 1 membrane).
In this way, differences in time exposure leading to different protein levels
were observed and corrected for. Standard deviations were also corrected by
using the same factor.
Immunohistochemistry. Immunohistochemistry was performed as previously described (3, 4). Studies were done on serial sections of control and shunted ovine lung using rabbit anti-oVEGF sera (BioSynthesis) and rabbit polyclonal anti-Flt-1 or goat polyclonal anti-KDR/Flk-1 (Santa Cruz Laboratories). Frozen tissue sections (7 µm) were thawed at room temperature. Samples were fixed for 10 min in cold acetone and then washed 3x with PBS. To eliminate nonspecific binding of the primary antiserum to tissue proteins, tissue sections were incubated with 1% horse serum in PBS (blocking solution) for 1 h. Tissue sections were then incubated with anti-oVEGF (1:100), anti-human KDR/Flk-1, or Flt-1 (5 µg/ml), in the presence of monoclonal smooth muscle cell-actin antibody (1:400, Sigma) in blocking solution at 4°C overnight. After three washes with PBS x 5 min, samples were visualized with Rhodamine Red-X goat anti-rabbit and Oregon Green 488 goat anti-mouse secondary antibodies (Molecular Probes) at a concentration of 1:400 in blocking solution for 45 min at room temperature. After three further washes with PBS, an anti-fading solution was added, and samples were visualized by fluorescence microscopy. For each tissue section, a parallel experiment was carried out in which the primary antibody was omitted. This served as the negative control. A minimum of three different sets of control and shunt lung tissues were prepared and examined. Because it is difficult to utilize immunohistochemistry for qualitative measurements on protein expression, we used this technique only to determine protein localization and to determine whether there were differences between shunt and control lambs in the numbers of vessels expressing VEGF, KDR/Flk-1, and Flt-1. To carry out this procedure, small muscularized pulmonary arteries were visualized next to airways, and at least 30 vessels (representing at least 10 fields) were counted as positively immunoreactive or nonimmunoreactive for a particular protein. The number of vessels immunoreactive for each protein as a proportion of the total vessels counted was determined. Results were then calculated as the average number of positively stained vessels ± SD (n = at least 3 different lambs from each age group), and statistical significance was calculated as described below.
Determination of circulating plasma VEGF levels. Because of
inherent differences between human VEGF and oVEGF, the commercially available
ELISA does not adequately detect oVEGF. Thus we developed a competitive ELISA
that specifically detects oVEGF. This ELISA uses a rabbit polyclonal antibody
against oVEGF that was synthesized using the sequence corresponding to
oVEGF120 (NH2-GCRIKPHQSQHIGEMSFLQHNK-COOH) as the
capture antibody (10 ng/ml in PBS). The methodology for measuring oVEGF is as
follows: a 96-well plate (Costar) is coated with 100 µl of capture antibody
at room temperature overnight. The plate is then washed three times with 0.05%
Tween in PBS and blocked with 300 µl/well of 5% sucrose and 1% BSA in PBS
for 1 h. The blocking solution is then aspirated and the plates washed as in
PBS/Tween. For the assay, plasma (150 µl) or various dilutions of standard
oVEGF (0.03–2 µg) are added together with biotinylated oVEGF (50
µl at 200 ng/ml) and allowed to bind to the capture antibody for 2 h at
room temperature. The wells are then aspirated and washed in PBS/Tween, and
streptavidin-horseradish peroxidase (100 µl, R&D Systems) is added for
20 min. The wells are then aspirated and washed, and a detection solution for
biotin (100 µl of tetramethylbenzidine, Sigma) is used to detect bound
biotinylated oVEGF. After 20 min, a stop solution of 2N
H2SO4 (50 µl) is used, and the plate is read at 450
nm. Unknown values for VEGF are then calculated with the aid of Tablecurve
software using the standard curve for known amounts of oVEGF. Cross-reaction
with human VEGF was found to be 20%, <1% with transforming growth factor
(TGF)-1 and nerve growth factor, and <5% with basic fibroblast growth
factor (bFGF).
Data analysis. Quantitation of protein expression was performed using a Kodak Image Station 440CF and KDS1D imaging software. This allows a pixel density from 1–103 instead of 256 gray scale of autoradiographic film. The response is linear within this range.
In all experiments, means ± SE were calculated, and comparisons between control and shunted lambs were made by ANOVA. When differences were present among study groups, Student-Newman-Keuls post hoc testing was performed. P < 0.05 was considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
VEGF signals through two tyrosine kinase membrane receptors: KDR/Flk-1 and Flt-1. Therefore, we analyzed the changes in protein expression of KDR/Flk-1 by Western blotting and immunohistochemistry. Its pattern of expression in the lungs of control lambs correlated well with that of VEGF where expression was higher at 1 day, then dropped to 25% at 1 wk, then 9% at 4 wk, and finally 3% at 8 wk of age compared with 1-day average values (Fig. 3, P < 0.05 relative to day 1 values). Relative to average values of control lambs, KDR/Flk-1 protein expression was significantly increased in the lungs of shunt animals at 1 wk and 4 wk of age (397% and 88%, respectively, P < 0.05, Fig. 3) and nonsignificantly decreased at 8 wk of age (57% of control values, Fig. 3). This is of interest since the increase in this receptor preceded the increase in VEGF expression. It is important to note that although VEGF expression in the shunt model showed a biphasic pattern (with 2 peaks of expression), KDR/Flk-1 expression in the shunt showed a delayed decline in values (Figs. 1 and 3). Immunohistochemistry studies showed that KDR/Flk-1 was present exclusively in the endothelium of small vessels (diameter <200 µm) and in capillaries (diameter <10 µm) of both control and shunted lambs (Fig. 4). Compared with control samples, shunt lung sections showed a nonsignificant increase in KDR/Flk-1 expression in the endothelium of small arteries at 1 wk (62.6 ± 11.6% positively stained vessels in the shunt compared with 29.6 ± 7.9% in control, P = 0.087). At 4 wk of age, shunt lung sections showed a significant increase in KDR/Flk-1 expression in the endothelium of small arteries (52.3 ± 10.2% stained vessels in the shunt compared with 16 ± 1.2% in control, P < 0.05; Fig. 4, A–H). In addition, there were more capillaries positively stained with KDR/Flk-1 in the 4-wk-old samples compared with control samples (Fig. 4, A–H).
|
|
In contrast with the normal developmental pattern of VEGF and KDR/Flk-1 expression, Flt-1 (VEGF-R1) protein expression in the normal lamb increased with age: 1-, 4-, and 8-wk-old average protein levels were 58, 49, and 174% higher than 1-day-old controls, respectively (Fig. 5). Compared with control values, average protein levels of Flt-1 in the shunt model were significantly elevated only at 4 wk of age (157% higher than control values, P < 0.05, Fig. 5). However, Flt-1 expression was nonsignificantly elevated at 1 and 8 wk of age (23 and 27% higher than control values, respectively, Fig. 5). Immunohistochemical analysis demonstrated that Flt-1 is abundantly expressed in the endothelium of pulmonary vessels (both veins and arteries) and of capillaries, and expression was found to be highest in both 8-wk-old control and shunted lambs. In contrast with the normal developmental pattern, expression of Flt-1 was elevated in medium- to small-sized pulmonary vessels in the 4-wk-old shunt (63.4 ± 6.7% stained vessels in the shunt compared with 30.7 ± 3% in controls, P < 0.05) but was similar to control lambs in microvessels (Fig. 6, A–H).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Several clinical and in vivo studies have shown a transient increase in local and circulating VEGF levels in models of increased blood flow, as occurs in response to increased exercise, pregnancy, and induced bradycardia (24, 40). In addition, increased local expression of VEGF and KDR/Flk-1 has been observed in vascular lesions occurring in various pulmonary hypertensive disorders (23, 39). For instance, increased VEGF expression has been observed in the tracheal aspirates and type II pneumocytes of neonates with PPHN (23). Similarly, VEGF levels are increased in the lungs of newborns with congenital diaphragmatic hernia and pulmonary hypertension (34) and in the lungs of adults with advanced pulmonary vascular disease secondary to congenital heart disease (13, 39). Our studies, using a model of increased pulmonary blood flow, suggest that selective upregulation of VEGF and its receptors in small pulmonary arteries indicates that dysregulation of this angiogenesis leads to altered endothelial behavior. In our studies, we were unable to observe a significant increase in VEGF circulating levels in the shunt compared with control values. This suggests that changes in the expression of VEGF may occur selectively in the pulmonary vessel walls of small arteries that become remodeled. Similarly, other studies have observed a selective upregulation of VEGF and other angiogenic factors in pulmonary vascular lesions with unchanging circulating levels of these growth factors, as in the plexiform lesions of severe primary pulmonary hypertension (13, 39).
We also observed increased expression of VEGF in the smooth muscle layer of small pulmonary arteries, in particular those that become remodeled, together with an increased expression of its receptors KDR/Flk-1 and Flt-1 in adjacent endothelium. Of interest is the fact that we observed a sequential increase in VEGF receptors, in which KDR/Flk-1 was increased at earlier stages (1–4 wk) and Flt-1 showed increases at later stages (4 and 8 wk of age). On VEGF binding, KDR/Flk-1 becomes rapidly phosphorylated at tyrosine residues, and this leads to endothelial mitogenesis, migration, and changes in cell morphology (degradation of stress fibers) (30). Therefore, KDR/Flk-1 mediates most VEGF angiogenic effects, thereby showing an important role in the activation phase of angiogenesis (2, 29).
Flt-1 has been implicated in upregulated endothelial expression of tissue
and urokinase plasminogen activator expression and plasminogen activator
inhibitor-1 (10,
20); these processes are
needed for vessel maturation and vessel wall integrity. Flt-1 has also been
reported to be expressed in vascular smooth muscle cells where it enhances
matrix metalloproteinase expression
(10,
29). Because of its higher
binding affinity with VEGF, Flt-1 has been suggested to sequester VEGF to
prevent its binding to KDR/Flk-1 and, therefore, act in the resolution phase
of angiogenesis (20). However,
studies on abnormal angiogenic processes indicate that Flt-1 can also mediate
positive angiogenic responses of VEGF, since it occurs in carcinogenic
processes (16). Our
observations on the initial increase in KDR/Flk-1 levels followed by a latter
increase in Flt-1 in the shunt model correlates with our previous observations
on increased blood vessel number at 4 wk of age and a subsequent decrease to
normal values at 8 wk of age. Moreover, our data suggest that increased
pulmonary blood flow, as occurs in our shunt model, dysregulates the process
of angiogenesis by upregulating VEGF and its receptors. However, our data also
indicate that these effects are temporal, with the increases in Flt-1 possibly
being involved in the resolution of these angiogenic events. Our data also
indicate that Flt-1 upregulation in the shunt at 4 wk of age correlates with
VEGF upregulation. This is of interest since both Flt-1 and VEGF
transcriptional regulation occur via similar transcription factors, i.e.,
hypoxia-inducible factor 1
and activator protein-1
(14).
The mechanisms by which increased pulmonary blood flow increase VEGF
expression remain unknown. It has been hypothesized that the increase in
angiogenic growth factors TGF-1 and VEGF observed in models of increased
blood flow is due to an increase in biomechanical forces, i.e., cyclic stretch
and laminar shear stress (15,
24,
41). In accordance with this
theory, various in vitro results have shown that cyclic stretch and laminar
shear stress increase VEGF expression in smooth muscle cells and
cardiomyocytes in a time-dependent fashion
(15,
24,
31,
37,
41). Many signaling molecules
have been proposed to mediate this effect, including indirect proangiogenic
molecules such as TGF-1, IL-1, and PDGF
(7). We have previously shown
an increased expression of TGF-1 and its proangiogenic receptors in our
shunt model.
Our previous data indicate that TGF-1 is upregulated as early as 1 wk
of age in the lungs of shunted lambs, preceding the increase in VEGF
expression. Furthermore, the data presented here indicate that increased VEGF
expression persists after TGF-1 expression returns to control levels,
suggesting that TGF-1 regulates the increases in VEGF observed at later
ages. Moreover, these molecular changes correlated with structural changes.
Together, these data suggest a role for coordinated expression of TGF-1
and VEGF in the alterations of pulmonary vascular morphology induced by
increased pulmonary blood flow secondary to congenital heart disease.
Alterations in pulmonary vascular remodeling and growth are a major source of morbidity and mortality for children and adults with congenital heart disease. Although early surgical correction of many defects has significantly reduced the incidence of irreversible pulmonary vascular disease, reversible remodeling may still be associated with altered reactivity that results in perioperative morbidity. In addition, mild pulmonary vascular remodeling in infants with single ventricle physiology and diminished pulmonary vascular growth in infants with pulmonary atresia may eliminate corrective surgical options. Therefore, the status of the pulmonary vasculature is often the principal determinant of outcome in congenital heart disease. Although the morphology is well described, the mechanisms of abnormal vascular growth are not well understood. In the present study, we describe novel alterations in VEGF expression in an animal model of congenital heart disease with increased pulmonary blood flow. A better understanding of these and other mechanisms could profoundly affect the timing and feasibility of surgical and medical treatments for congenital heart disease and, therefore, warrants further investigations.
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
in fibroblastic and epithelial cells. J Biol
Chem 269:
6271-6274, 1994.1 in patients
with atrial fibrillation undergoing defibrillation therapy. Jpn
Heart J 41:
27-32, 2000.[Medline]
.
Am J Physiol Heart Circ Physiol
280: H909-H917,
2001.This article has been cited by other articles:
|
|
 |
|
  L. Ray, M. Mathieu, P. Jespers, I. Hadad, M. Mahmoudabady, A. Pensis, S. Motte, I. R. Peters, R. Naeije, and K. McEntee Early increase in pulmonary vascular reactivity with overexpression of endothelin-1 and vascular endothelial growth factor in canine experimental heart failure Exp Physiol, March 1, 2008; 93(3): 434 - 442. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Black, J. M. DeVol, and S. Wedgwood Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation Am J Physiol Cell Physiol, January 1, 2008; 294(1): C345 - C354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T.R. Roofthooft, K. A. Bergman, T. W. Waterbolk, T. Ebels, B. Bartelds, and R. M.F. Berger Persistent Pulmonary Hypertension of the Newborn With Transposition of the Great Arteries Ann. Thorac. Surg., April 1, 2007; 83(4): 1446 - 1450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Majka, K. Fox, B. McGuire, J. Crossno Jr., P. McGuire, and A. Izzo Pleiotropic role of VEGF-A in regulating fetal pulmonary mesenchymal cell turnover Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1183 - L1192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-F. Lam, T. E. Peterson, A. J. Croatt, K. A. Nath, and Z. S. Katusic Functional adaptation and remodeling of pulmonary artery in flow-induced pulmonary hypertension Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2334 - H2341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Mata-Greenwood, A. Grobe, S. Kumar, Y. Noskina, and S. M. Black Cyclic stretch increases VEGF expression in pulmonary arterial smooth muscle cells via TGF-{beta}1 and reactive oxygen species: a requirement for NAD(P)H oxidase Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L288 - L289. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
P
<0.05 vs. 1 day control. C: representative RNase protection assays
are shown from a cRNA probe for ovine VEGF that was hybridized overnight to 50
µg of total lung RNA prepared from 1-day-old and 1-, 4-, and 8-wk-old lambs
(1 control and 1 shunt from each age). VEGF mRNA expression was increased in
shunted lambs only at 4 and 8 wk. There were no protected fragments detected
in the lanes where the probe was hybridized without RNA (PA) or in the
presence of tRNA. VEGF is undigested probe. A cRNA for ovine 18S was also
hybridized to serve as a control for RNA loading. D: densitometric
values for relative VEGF mRNA (normalized to 18S mRNA and to control values)
from 5 control and 5 shunted lambs at each age. In shunted lambs, relative
VEGF mRNA increased by 147% at 4 wk and 98% at 8 wk (P <0.05).
Values are means ± SE. *P <0.05 control vs.
shunt.
