PDGF Receptor Protein Tyrosine Kinase Expression in the Balloon-Injured Rat Carotid Artery

Abstract

Platelet-derived growth factor (PDGF) receptor gene expression has previously been demonstrated in balloon-injured rat carotid arteries to be regulated during repair of carotid injury. In the present study we showed that PDGF receptor protein expression and phosphorylation are changed over time after carotid artery injury. In control and 2-day-postinjury vessels, expression of PDGF alpha receptor protein was readily detectable, whereas PDGF beta receptor expression appeared very low. Between 2 and 7 days postinjury, a time interval previously shown to correspond with smooth muscle cell migration followed by the appearance of a neointima, PDGF alpha receptor expression had increased only slightly, to roughly 35% above control levels, and was maximal by day 7 postinjury, whereas PDGF beta receptor expression had doubled. From 7 to 14 days after carotid injury, intimal area was greatly increased and was associated with a further increase in PDGF beta receptor protein expression and receptor phosphorylation to a maximum between days 10 and 12. In contrast, PDGF alpha receptor expression had decreased slightly during this time interval. Moreover, phosphorylation of PDGF alpha receptors was barely detectable and did not change over the time course of injury. From 14 to 28 days after injury, intimal area was increased only slightly, whereas PDGF beta receptor protein and phosphorylation levels had diminished to roughly half of the 10-day injury values. In addition, the increase in PDGF beta receptor protein expression and tyrosine phosphorylation observed over the time of injury were also associated with a corresponding increase in the association of phosphatidylinositol 3' kinase (PI-3 kinase) with phosphorylated PDGF beta receptors. These findings show that balloon injury to rat carotid arteries results in temporally related changes in the expression of PDGF receptors and their state of tyrosine phosphorylation. Furthermore, tyrosine phosphorylation of PDGF beta receptors in the balloon-injured rat carotid artery in vivo resulted in the association of PI-3 kinase. These are important new findings, which add to our knowledge concerning the role and activity of PDGF receptors in the formation of a neointima.

Local Delivery of Platelet-Derived Growth Factor Receptor–Specific Tyrphostin Inhibits Neointimal Formation in Rats

“…All of these results correspond well with our finding of moderately upregulated PDGFR␤ expression on day 3 after injury and a marked increase in receptor expression after 2 weeks ( Figure 5A). Our preliminary experiments after immunoprecipitation of PDGFR␤ validated that the band migrating at 190 kDa is PDGFR␤, as was shown by Panek et al 40 and by the findings in the present study (190 kDa and the similar band location after immunoblotting with anti-PDGFR␤ and anti-phosphotyrosine antibodies; compare Figure 5A and 5B).…”

“…We, 25 as well as others, 24,40 have previously shown that balloon injury upregulates overall tyrosine phosphorylation in vascular SMCs. In accordance with these early findings, we have shown in the present study (Figure 5B) that tyrosine phosphorylation of PDGFR␤ (190 kDa) and of several other proteins was increased.…”

“…These results were confirmed recently by Sirois et al, 50 who showed that balloon injury enhanced PDGFR␤ expression in SMCs of the media and nascent neointima after 2 weeks. Panek et al 40 have shown a very weak basal expression of PDGFR␤ on SMCs of intact rat carotid arteries that was not significantly changed 2 days after injury. The respective band was markedly enhanced in blots from arteries of rats euthanized 7 to 12 days after injury and then decreased at later time points.…”

“…Other important proteins demonstrating an increase in phosphotyrosine content after PDGF-BB or FCS treatment in the present study could be (1) the focal adhesion kinase (FAK, 125 kDa), (2) the regulatory subunit of phosphatidylinositol-3Ј kinase (85 kDa), (3) the c-Src (60 kDa), or (4) the mitogen-activated protein (MAP) kinase isoforms (42/44 kDa). FAK, 39 phosphatidylinositol-3Ј kinase, 40 and MAP kinase (ERK 1/2) 41 were reported to be rapidly phosphorylated on distinct tyrosine residues on PDGF-BB stimulation. The inhibitory effect of AG-1295 on PDGF-BB-induced tyrosine phosphorylation of PDGFR␤ was reversible, in view of the fact that complete recovery was observed after washout of the inhibitor ( Figure 3C).…”

The Role of Alpha and Beta Platelet-Derived Growth Factor Receptor in the Vascular Response to Injury in Nonhuman Primates

“…2,4,16,26,29 -35 Although alpha PDGFR was not detected in baboon arteries in the present study, low levels of this receptor have been detected in the media of the rat carotid artery and in the neointima of healing vascular grafts in baboons. 16,29,32,33,35 Alpha PDGFR expression by medial SMC could be functionally important because PDGF A chain, which only interacts with the alpha receptor, has been routinely observed in atherosclerotic plaques and in medial cells after injury. 1,2,16,26,29 To address the relative importance of alpha and beta PDGFR in the vascular response to injury, we used the isotype-matched neutralizing mAbs 2H7C5 and 2A1E2 directed against alpha and beta PDGFRs, respectively.…”