Kevin Krasinski

RECIPROCAL RELATION BETWEEN VEGF AND NO IN THE REGULATION OF ENDOTHELIAL INTEGRITY

Balloon angioplasty disrupts the protective endothelial lining of the arterial wall, rendering arteries susceptible to thrombosis and intimal thickening. We show here that vascular endothelial growth factor (VEGF), an endothelial cell mitogen, is upregulated in medial smooth muscle cells of the arterial wall in response to balloon injury. Both protein kinase C (PKC) and tyrosine kinase pp60src mediate augmented VEGF expression. In contrast, nitric oxide (NO) donors inhibit PKC-induced VEGF upregulation by interfering with binding of the transcription factor activator protein-1 (AP-1) to the VEGF promoter. Inhibition of VEGF promoter activation suggests that NO secreted by a restored endothelium functions as the negative feedback mechanism that downregulates VEGF expression to basal levels. Administration of a neutralizing VEGF antibody impaired reendothelialization following balloon injury performed in vivo. These findings establish a reciprocal relation between VEGF and NO in the endogenous regulation of endothelial integrity following arterial injury.

Evidence for the Rapid Onset of Apoptosis in Medial Smooth Muscle Cells After Balloon Injury

BACKGROUND Vascular myocyte apoptotic cell death has been reported in human atherectomy and endarterectomy specimens and for neointimal smooth muscle cells (SMCs) in balloon-injured rat carotid arteries between 7 and 30 days after injury. However, the immediate effect of balloon injury on medial SMC viability has not been examined. METHODS AND RESULTS Rat carotid arteries were harvested at the time of balloon injury (T = 0) and at 0.5, 1, 2, and 4 hours after injury. Uninjured vessels or vessels harvested at the time of injury (T = 0) did not display evidence of apoptosis. However, as early as 30 minutes after injury, 70% of medial SMCs appeared apoptotic by TdT-mediated dUTP nick end labeling (TUNEL) analysis and by the appearance of condensed chromatin. High frequencies of TUNEL-positive cells were also observed at 1 and 2 hours after injury but not at 4 hours. Transmission electron microscopy revealed many cells with morphological characteristics of apoptosis in the injured sections. A marked decrease in bcl-X expression was detected in the most luminal layers of the media. To corroborate these findings in a second animal model, rabbit external iliac arteries were analyzed after balloon angioplasty. Apoptotic cell death was evident in rabbit arteries at 30 minutes and at 4 hours after injury. CONCLUSIONS As early as 30 minutes after balloon injury, myocytes appear to undergo apoptotic cell death at a high frequency as shown by TUNEL staining, chromatin condensation, and the appearance of morphological features in electron micrographs. The induction of apoptosis coincides with a marked downregulation of bcl-X expression.

Estradiol Accelerates Functional Endothelial Recovery After Arterial Injury

BACKGROUND The mechanisms of the established atheroprotective effects of estrogen have not been entirely clarified. Recent data suggest that agents that hasten the recovery of the endothelium after denuding injury will deter the development of neointimal lesions. Because estrogen has been shown to exert angiogenic effects in vitro and in vivo, we performed a series of experiments to evaluate whether estrogen was capable of accelerating reendothelialization. METHODS AND RESULTS Ovariectomized Sprague-Dawley rats received estrogen replacement therapy in the form of subcutaneously implanted pellets designed to release 1.5 or 5.0 mg 17 beta-estradiol over 30 days. Deendothelializing balloon injury was performed 1 week after pellet implantation, and animals were euthanatized after 1 week for evaluation of reendothelialization (Evans blue staining) or 2 weeks for evaluation of reendothelialization and neointimal formation. At both time points, the use of estradiol caused a dose-dependent increase in reendothelialization, which was measured as absolute area and percentage of area that is reendothelialized. Estradiol accelerated functional endothelial recovery, manifested as an increase in nitric oxide production. Neointimal thickening was also shown to be inhibited in a dose-dependent fashion. CONCLUSIONS Estrogen accelerates functional endothelial recovery after barotraumatic deendothelializing injury. These findings, along with the recent demonstration of estrogen receptor expression by endothelial cells, suggest that the antiatherogenic action of estrogen may be mediated in part through direct effects on endothelial cells.

Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27(KIP1), an inhibitor of neointima formation in the rat carotid artery.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhibitory protein p27(KIP1) (p27). Ectopic overexpression of p27 in serum-stimulated VSMCs resulted in the inhibition of cdk2 activity and repression of cyclin A promoter activity. Collectively, these findings indicate that p27 may contribute to VSMC growth arrest in vitro. Using the rat carotid model of balloon angioplasty, a marked upregulation of p27 was observed in injured arteries. High levels of p27 expression in the media and neointima correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27 in balloon-injured arteries attenuated neointimal lesion formation. Thus, the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty.

Antibody Blockade of Thrombospondin Accelerates Reendothelialization and Reduces Neointima Formation in Balloon-Injured Rat Carotid Artery

BACKGROUND Remodeling of the extracellular matrix plays an important role during the pathogenesis of atherosclerosis and restenosis. The matrix glycoprotein thrombospondin-1 (TSP1) inhibits endothelial cell proliferation and migration in vitro. In contrast, TSP1 facilitates the growth and migration of cultured vascular smooth muscle cells. Accordingly, we investigated the hypothesis that administration of anti-TSP1 antibody could facilitate reendothelialization and inhibit neointimal thickening in balloon-injured rat carotid artery. METHODS AND RESULTS Sprague-Dawley rats were subjected to left common carotid artery denudation, after which arteries were treated with C6.7 anti-TSP1 or control antibody. Evans blue dye staining 2 weeks after injury disclosed significantly increased reendothelialization in arteries treated with C6.7 antibody compared with the control group, and this effect was associated with increased number of proliferating cell nuclear antigen-positive endothelial cells. In contrast, treatment with C6.7 antibody decreased the number of proliferating cell nuclear antigen-positive vascular smooth muscle cells in the injured arterial wall. Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries receiving C6.7 antibody versus the control group at both the 2-week and 4-week time points. CONCLUSIONS Intra-arterial delivery of antibody against TSP1 facilitated reendothelialization and reduced neointimal lesion formation after balloon denudation.

Role of c-fos and E2F in the induction of cyclin A transcription and vascular smooth muscle cell proliferation.

Excessive proliferation of vascular smooth muscle cells (VSMCs) contributes to vessel renarrowing after angioplasty. Here we investigated the transcriptional regulation of the cyclin A gene, a key positive regulator of S phase that is induced after angioplasty. We show that Ras-dependent mitogenic signaling is essential for the normal stimulation of cyclin A promoter activity and DNA synthesis in VSMCs. Overexpression of the AP-1 transcription factor c-fos can circumvent this requirement via interaction with the cAMP-responsive element (CRE) in the cyclin A promoter. Moreover, c-fos overexpression in serum-starved VSMCs results in the induction of cyclin A promoter activity in a CRE-dependent manner, and increased binding of endogenous c-fos protein to the cyclin A CRE precedes the onset of DNA replication in VSMCs induced by serum in vitro and by angioplasty in vivo. We also show that E2F function is essential for both serum- and c-fos-dependent induction of cyclin A expression. Taken together, these findings suggest that c-fos and E2F are important components of the signaling cascade that link Ras activity to cyclin A transcription in VSMCs. These studies illustrate a novel link between the transcriptional and cell cycle machinery that may be relevant to the pathogenesis of vascular proliferative disorders.

Adenoviral Constructs Encoding Phosphorylation-Competent Full-length and Truncated Forms of the Human Retinoblastoma Protein Inhibit Myocyte Proliferation and Neointima Formation

BACKGROUND The retinoblastoma (Rb) protein is a key cell-cycle regulator that controls entry into the S phase by modulating the activity of the E2F transcription factor. We analyzed the effects of full-length phosphorylation-competent and a mutant truncated form of human Rb for their effects on vascular smooth muscle cell (VSMC) proliferation and neointima formation. METHODS AND RESULTS A number of mutant forms, both phosphorylation competent and incompetent, of human Rb protein were evaluated for their ability to inhibit E2F activity. The results of these assays indicated that a phosphorylation competent, amino-terminal-truncated Rb protein (Rb56) was a particularly potent inhibitor of E2F-mediated transcription relative to the full-length Rb construct (Rb110). Adenoviral constructs containing Rb56 or Rb110 expressed their respective Rb forms in VSMCs, as determined by Western immunoblot analysis, and were similar in their abilities to arrest the cell cycle, as determined by assays of 3H-thymidine incorporation and by flow cytometric analyses. When examined for their effect on neointima formation after balloon injury of the rat carotid artery, both full-length and truncated forms of Rb inhibited formation of this VSMC-derived lesion. CONCLUSIONS These analyses revealed that the maintenance of high levels of phosphorylation-competent human Rb, either full-length or truncated forms, in VSMCs is an effective method of modulating the extent of intimal hyperplasia that occurs after balloon-induced vascular injury.

Adenovirus-encoded hammerhead ribozyme to Bcl-2 inhibits neointimal hyperplasia and induces vascular smooth muscle cell apoptosis

OBJECTIVE The balance between apoptosis and cell proliferation is vital for cellular homeostasis, yet little is known about the mechanisms that coordinate these two cell fates, particularly in the vessel wall. It is well established that the members of Bcl-2-gene family are regulators of apoptosis, but their role in cellular proliferation is less clear. METHODS We analyzed the effects of disrupting Bcl-2 expression in vascular smooth muscle cells (VSMCs) by adenoviral-mediated delivery of a hammerhead ribozyme against bcl-2 mRNA (Ad-Rbz-Bcl-2). RESULTS Forced ablation of Bcl-2 in balloon-injured rat carotid arteries reduced cell number and inhibited neointimal hyperplasia. In vitro, VSMCs transduced with the Ad-Rbz-Bcl-2 underwent apoptosis as indicated by a reduction in cell number and DNA fragmentation. Ad-Rbz-Bcl-2-transduced cells also exhibited aberrations in both G1- and S-phases of the cell cycle. However, forced perturbations in cell cycle activity by serum-stimulation or treatment with chemical inhibitors did not affect Ad-Rbz-Bcl-2-induced cell death, indicating that these cell cycle changes are not essential for apoptosis. CONCLUSION These data show that physiological levels of Bcl-2 are essential for VSMC viability and that ablation of Bcl-2 alters cell cycle activity through the execution of the apoptotic process.

Restoration of E2F Expression Rescues Vascular Endothelial Cells From Tumor Necrosis Factor-α–Induced Apoptosis

BACKGROUND Normally, quiescent endothelial cells (EC) line the inner surface of arteries and protect against thrombosis and neointimal growth. A variety of noxious stimuli, including balloon angioplasty, may compromise EC integrity, thereby initiating proliferation and triggering the local release of cytokines, including tumor necrosis factor-alpha (TNF-alpha). METHODS AND RESULTS In vivo blockade of TNF-alpha using a soluble receptor molecule results in accelerated reendothelialization at sites of balloon angioplasty, suggesting an important physiological role of TNF-alpha in attenuating regrowth of endothelium after balloon angioplasty. Our studies reveal that TNF-alpha, an apoptosis-inducing cytokine, induces G1 cell-cycle arrest in proliferating EC. Quiescent EC are relatively immune to TNF-induced apoptosis versus proliferating EC, which display repression of the E2F transcription factor coincident with TNF-induced apoptosis and cell-cycle arrest. We also show that in this setting, E2F overexpression exerts a survival effect in proliferating EC and restores cell-cycle progression, in direct contrast to results of prior reports, which revealed that deregulated expression of E2F in normally cycling cells induces apoptosis. CONCLUSIONS These data demonstrate that TNF-induced apoptosis is highly dependent on cell-cycle activity and that E2F can function as survival factor under certain conditions.

Engineering the Response to Vascular Injury: Divergent Effects of Deregulated E2F1 Expression on Vascular Smooth Muscle Cells and Endothelial Cells Result in Endothelial Recovery and Inhibition of Neointimal Growth

&NA; Tumor necrosis factor‐&agr; (TNF‐&agr;) is expressed locally in the vessel wall after angioplasty and induces growth arrest and apoptosis in endothelial cells (ECs), thereby delaying reendothelialization. Prior studies have shown that direct antagonism of TNF‐&agr;, using a systemically administered soluble receptor, can enhance endothelial recovery and reduce neointimal thickening. These studies have also shown that downregulation of the transcription factor E2F1 was a key mechanism of TNFs effect on ECs. We now show that Ad‐E2F1 overexpression at sites of balloon injury accelerates functional endothelial recovery, consistent with the prior in vitro findings. Moreover these studies also reveal divergent effects of TNF‐&agr; and overexpression of E2F1 on ECs versus VSMCs. TNF‐&agr; exposure of VSMCs had no affect on proliferation or apoptosis, in contrast to the effect seen in ECs. In Ad‐E2F1‐transduced VSMCs, however, TNF‐&agr;‐induced marked apoptosis in contrast to the survival effect seen in ECs. Finally, these studies suggest that differential activation of NF‐&kgr;B may play a key role in mediating these opposing effects. Nuclear translocation and transcriptional activity of NF‐&kgr;B was markedly attenuated in Ad‐E2F1‐transduced VSMCs, whereas it remained active in similarly treated ECs when the cells were exposed to TNF‐&agr;. These studies reveal that overexpression of Ad‐E2F1 primes VSMCs to TNF‐&agr;‐induced apoptosis. Furthermore, E2F1 potentiates VSMC death by blocking antiapoptotic signaling pathway through inhibition of NF‐&kgr;B activation. The divergent responses of VSMCs and ECs to E2F1 overexpression provide unique therapeutic possibilities: simultaneously targeting the cell cycle of two different cell types, within same tissue microenvironment resulting in opposite and biologically complimentary effects. (Circ Res. 2003;93:162‐169.)