Expression of VEGF and its receptors Flt-1 and Flk-1/KDR is altered in lambs with increased pulmonary blood flow and pulmonary hypertension

Eugenia Mata-Greenwood,1 Barbara Meyrick,2 Scott J. Soifer,3,4 Jeffrey R. Fineman,3,4 and Stephen M. Black1,5

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
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Utilizing in utero aortopulmonary vascular graft placement, we developed a lamb model of congenital heart disease and increased pulmonary blood flow. We showed previously that these lambs have increased pulmonary vessel number at 4 wk of age. To determine whether this was associated with alterations in VEGF signaling, we investigated vascular changes in expression of VEGF and its receptors, Flt-1 and KDR/Flk-1, in the lungs of shunted and age-matched control lambs during the first 8 wk of life. Western blot analysis demonstrated that VEGF, Flt-1, and KDR/Flk-1 expression was higher in shunted lambs. VEGF and Flt-1 expression was increased at 4 and 8 wk of age (P <0.05). However, KDR/Flk-1 expression was higher in shunted lambs only at 1 and 4 wk of age (P <0.05). Immunohistochemical analysis demonstrated that, in control and shunted lambs, VEGF localized to the smooth muscle layer of vessels and airways and to the pulmonary epithelium while increased VEGF expression was localized to the smooth muscle layer of thickened media in remodeled vessels in shunted lambs. VEGF receptors were localized exclusively in the endothelium of pulmonary vessels. Flt-1 was increased in the endothelium of small pulmonary arteries in shunted animals at 4 and 8 wk of age, whereas KDR/Flk-1 was increased in small pulmonary arteries at 1 and 4 wk of age. Our data suggest that increased pulmonary blood flow upregulates expression of VEGF and its receptors, and this may be important in development of the vascular remodeling in shunted lambs.

vascular graft placement; shunted lambs


VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) is a cell-specific angiogenic and vasculogenic mediator (11, 29). It is observed ubiquitously at sites of angiogenesis, and its levels correlate closely with the spatial and temporal events of blood vessel growth (36). Some of the most prominent roles of VEGF include induction of endothelial cell proliferation and migration, induction of endothelial expression of proteases, stimulation of microvascular leakage, and increase in nascent endothelial cell survival (9, 11, 29). These effects are mediated by two transmembrane tyrosine kinase endothelial-specific receptors: fms-like tyrosine kinase-1 (Flt-1) and kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) (9, 11, 29). Molecular cloning of the cDNAs for VEGF have revealed that alternative mRNA splicing results in the generation of at least six isoforms that vary between 120 and 205 amino acids in length (19, 38).

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
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Surgical preparation and care. Forty mixed-breed, pregnant Western ewes (137–141 days gestation, term = 145 days) were operated on under sterile conditions as previously described (28). After spontaneous delivery of the lambs (2–7 days after surgery), antibiotics [106 units of penicillin G potassium and 25 mg of gentamicin sulfate intramuscularly (im)] were administered for 2 days. The lambs were weighed daily, and respiratory rate and heart rate were measured. Furosemide (1 mg/kg im) was administered daily, and elemental iron (50 mg im) was given weekly. At 1 day and at 1, 4, or 8 wk of age, the lambs were anesthetized with intravenous infusions of ketamine hydrochloride (~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 {beta}-actin. Each densitometric value was divided by its {beta}-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)-{beta}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
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of VEGF protein expression by immunoblotting in the lungs of normal lambs (controls) showed a progressive decline: 1-wk-olds expressed 0.44, 4-wk-olds expressed 0.25, and 8-wk-olds expressed 0.3 of day 1 values (Fig. 1). In the shunt model, VEGF protein expression was equivalent to controls at 1 day and 1 wk. However, VEGF expression was significantly increased in the peripheral lung tissue of shunted lamb lung at 4 and 8 wk of age (459% and 149%, P < 0.05, Fig. 1, A and B). Analysis of VEGF mRNA by RNase protection assays showed a similar pattern, where VEGF mRNA was significantly increased in shunted lambs at 4 and 8 wk of age (147% and 98% of controls, respectively, P < 0.05) but not at either 1 day or 1 wk of age (Fig. 1, C and D). Analysis of circulating levels of VEGF in plasma samples from 1- to 8-wk-old control and shunted lambs did not reveal any significant increases compared with controls (data not shown). Immunohistochemical analysis of VEGF in conjunction with smooth muscle actin revealed that VEGF was highly expressed in the epithelium and smooth muscle layer of airways from control and shunted lambs. However, 4- and 8-wk-old shunted lambs showed an increased VEGF expression in the smooth muscle layer of small pulmonary vessels, especially in those possessing a thickened media (Fig. 2, A–H). There was no major change in expression or localization of VEGF at 1 day and 1 wk between shunted and control lambs.



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Fig. 1. Western blot and RNase protection analyses for vascular endothelial growth factor (VEGF) in peripheral lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). A: representative Western blots are shown from protein extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each age), separated on a 4–20% SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against VEGF. VEGF protein expression was increased in shunted lambs only at 4 and 8 wk. The band shown for VEGF is 25 kDa and represents the monomeric form of VEGF. Also included on each gel was an internal control [peripheral lung extract (50 µg) prepared from a 1-day-old shunt]. Each membrane was also reprobed for {beta}-actin to normalize for differences in protein loading. Con, control; Sh, shunt. B: densitometric values for relative VEGF protein from 5 control and 5 shunted lambs at each age was determined as described in METHODS. In shunted lambs, relative VEGF protein is increased by 459% at 4 wk and 149% at 8 wk (P < 0.05). Values are means ± SE. *P <0.05 for control vs. shunt; {dagger}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.

 


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Fig. 2. VEGF protein expression in vivo in lung sections prepared from 1-day-old and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical localization of VEGF expression in the lung in vivo from 1-day-old (A and B), 1-wk-old (C and D), 4-wk-old (E and F), and 8-wk-old (G and H) lambs: control (A, C, E, G) and shunt (B, D, F, H). Polyclonal rabbit anti-VEGF antiserum and monoclonal mouse anti-smooth muscle cell-actin antibody were used to localize expression of VEGF. VEGF expression is shown in red, whereas smooth muscle cell-actin expression is shown in green. Colocalization is shown in yellow. Magnification is x200. Micrographs shown are representative of at least 3 different sets of twin matches (control and shunt). AW, airway; V, vessel. Smooth muscle layers from pulmonary arteries from 4- and 8-wk-old shunts, but not controls, show intense VEGF staining.

 

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).



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Fig. 3. Western blot analysis for kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) in peripheral lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). A: representative Western blots are shown from protein extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each age), separated on a 7.5% SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond membranes, and analyzed using a goat polyclonal anti-human KDR/Flk-1. KDR/Flk-1 protein expression was increased in shunted lambs only at 1 and 4 wk. Internal controls were used to compare relative amounts among the developmental ages. B: densitometric values for relative KDR/Flk-1 protein from 5 control and 5 shunted lambs at each age. In shunted lambs, relative KDR/Flk-1 protein is increased by 397% at 1 wk and 88% at 4 wk (P < 0.05). Values are means ± SE. *P <0.05 for control vs. shunt; {dagger}P < 0.05 vs. 1-day control.

 


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Fig. 4. KDR/Flk-1 protein expression in vivo in lung sections prepared from 1-day-old and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical localization of KDR/Flk-1 expression in the lung in vivo from 1-day-old (A and B), 1-wk-old (C and D), 4-wk-old (E and F), and 8-wk-old (G and H) lambs: control (A, C, E, G) and shunt (B, D, F, H). Polyclonal goat anti-KDR/Flk-1 antibody and monoclonal mouse anti-smooth muscle cell-actin antibody were used to localize expression of KDR/Flk-1. KDR/Flk-1 expression is shown in red, whereas smooth muscle cell-actin expression is shown in green. Colocalization is shown in yellow. Magnification is x200. Micrographs shown are representative of at least 3 different sets of twin matches (control and shunt). Arrows, pulmonary arteries. Pulmonary arteries from 1- and 4-wk-old shunts, but not controls, show intense KDR/Flk-1 staining in the endothelium of pulmonary arteries.

 

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).



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Fig. 5. Western blot analysis for fms-like tyrosine kinase (Flt-1) in peripheral lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). A: representative Western blots are shown from protein extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each age), separated on a 7.5% SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against Flt-1. The band shown for Flt-1 is 180 kDa as the 130-kDa band was nonspecific. Flt-1 protein expression was increased in shunted lambs only at 4 wk. B: average densitometric values for Flt-1/{beta}-actin protein from 5 control and 5 shunted lambs at each age. In shunted lambs, relative Flt-1 protein is increased by 157% at 4 wk (P < 0.05). Values are means ± SE. *P < 0.05 for control vs. shunt; {dagger}P < 0.05 vs. 1-day control.

 


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Fig. 6. Flt-1 protein expression in vivo in lung sections prepared from 1-day-old and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical localization of Flt-1 expression in the lung in vivo from 1-day-old (A and B), 1-wk-old (C and D), 4-wk-old (E and F), and 8-wk-old (G and H) lambs: control (A, C, E, G) and shunt (B, D, F, H). Polyclonal rabbit anti-Flt-1 antibody and monoclonal mouse anti-smooth muscle cell-actin antibody were used to localize expression of Flt-1. Flt-1 expression is shown in red, whereas smooth muscle cell-actin expression is shown in green. Colocalization is shown in yellow. Magnification is x200. Micrographs shown are representative of at least 3 different sets of twin matches (control and shunt). Arrows, pulmonary arteries. Four-week-old pulmonary arteries from shunt, but not controls, show intense Flt-1 staining.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In an effort to begin to unravel the molecular mechanisms involved in the process of vascular remodeling due to increased pulmonary blood flow and/or pressure, we investigated the expression of VEGF and its two main receptors, KDR/Flk-1 and Flt-1, in our lamb model of pulmonary hypertension with increased pulmonary blood flow. We observed that VEGF and its receptors are upregulated in the pulmonary circulation of shunted lambs compared with age-matched controls. The increase in VEGF expression observed in the shunt model correlated with an increase in blood vessel number, as we have reported previously (28). This observation is in accordance with the potent mitogenic effect of VEGF on endothelial cells (9, 11). The angiogenic process involves endothelial proliferation and migration, matrix degradation and invasion, and capillary/vessel morphogenesis (25). VEGF has been shown to participate in this process mainly in the initial steps of inducing endothelial proliferation and migrations (25). VEGF has been shown to synergize with bFGF, which has a role in endothelial cell reorganization and vessel morphogenesis, i.e., in late angiogenesis (8). Tissue invasion is achieved, in part, by an increase in extracellular matrix proteases like tissue plasminogen activator, urokinase plasminogen activator and its receptor, and plasmin (8, 25). VEGF upregulates the expression of these proteases at the transcriptional level (8). In addition, VEGF also possesses other roles involved in an adaptive response of the vascular system to an insult. For instance, VEGF has been shown to stimulate endothelial recovery from injury (1, 21) and to stimulate endothelial nitric oxide synthase expression and activity leading to vasodilation (35). Therefore, upregulation of VEGF might be an adaptive response to increased blood flow.

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{alpha} 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-{beta}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-{beta}1, IL-1, and PDGF (7). We have previously shown an increased expression of TGF-{beta}1 and its proangiogenic receptors in our shunt model.

Our previous data indicate that TGF-{beta}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-{beta}1 expression returns to control levels, suggesting that TGF-{beta}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-{beta}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
 

Address for reprint requests and other correspondence: S. M. Black, Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: steveblack{at}northwestern.edu).

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.


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