Zhengyu Luo

The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals

Recent studies suggest that statins can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. We show here that statins rapidly activate the protein kinase Akt/PKB in endothelial cells. Accordingly, simvastatin enhanced phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibited apoptosis and accelerated vascular structure formation in vitro in an Akt-dependent manner. Similar to vascular endothelial growth factor (VEGF) treatment, both simvastatin administration and enhanced Akt signaling in the endothelium promoted angiogenesis in ischemic limbs of normocholesterolemic rabbits. Therefore, activation of Akt represents a mechanism that can account for some of the beneficial side effects of statins, including the promotion of new blood vessel growth.

Acute modulation of endothelial Akt/PKB activity alters nitric oxide–dependent vasomotor activity in vivo

The serine/threonine protein kinase Akt (protein kinase B) phosphorylates endothelial cell nitric oxide synthase (eNOS) and enhances its ability to generate nitric oxide (NO). Because NO is an important regulator of vasomotor tone, we investigated whether Akt can regulate endothelium-dependent vasomotion in vivo using a rabbit femoral artery model of gene transfer. The endothelium of isolated femoral arteries was infected with replication-defective adenoviral constructs expressing beta-galactosidase, constitutively-active Akt (myr-Akt), or dominant-negative Akt (dn-Akt). Femoral arteries transduced with myr-Akt showed a significant increase in resting diameter and blood flow, as assessed by angiography and Doppler flow measurements, respectively. L-NAME, an eNOS inhibitor, blocked myr-Akt-mediated vasodilatation. In contrast, endothelium-dependent vasodilatation in response to acetylcholine was attenuated in vessels transduced with dn-Akt, although these vessels showed normal responses to nitroglycerin, an endothelium-independent vasodilator. Similarly, relaxation of murine aorta ex vivo in response to acetylcholine, but not nitroglycerin, was inhibited by transduction of dn-Akt to the endothelium. These data provide evidence that Akt functions as key regulator of vasomotor tone in vivo.

Akt Signaling Mediates Postnatal Heart Growth in Response to Insulin and Nutritional Status

Akt is a serine-threonine kinase that mediates a variety of cellular responses to external stimuli. During postnatal development, Akt signaling in the heart was up-regulated when the heart was rapidly growing and was down-regulated by caloric restriction, suggesting a role of Akt in nutrient-dependent regulation of cardiac growth. Consistent with this notion, reductions in Akt, 70-kDa S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1 phosphorylation were observed in mice with cardiac-specific deletion of insulin receptor gene, which exhibit a small heart phenotype. In contrast to wild type animals, caloric restriction in these mice had little effect on Akt phosphorylation in the heart. Furthermore, forced expression of Akt1 in these hearts restored 70-kDa S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 phosphorylation to normal levels and rescued the small heart phenotype. Collectively, these results indicate that Akt signaling mediates insulin-dependent physiological heart growth during postnatal development and suggest a mechanism by which heart size is coordinated with overall body size as the nutritional status of the organism is varied.

Myogenic Akt Signaling Regulates Blood Vessel Recruitment during Myofiber Growth

ABSTRACT Blood vessel recruitment is an important feature of normal tissue growth. Here, we examined the role of Akt signaling in coordinating angiogenesis with skeletal muscle hypertrophy. Hypertrophy of C2C12 myotubes in response to insulin-like growth factor 1 or insulin and dexamethasone resulted in a marked increase in the secretion of vascular endothelial growth factor (VEGF). Myofiber hypertrophy and hypertrophy-associated VEGF synthesis were specifically inhibited by the transduction of a dominant-negative mutant of the Akt1 serine-threonine protein kinase. Conversely, transduction of constitutively active Akt1 increased myofiber size and led to a robust induction of VEGF protein production. Akt-mediated control of VEGF expression occurred at the level of transcription, and the hypoxia-inducible factor 1 regulatory element was dispensable for this regulation. The activation of Akt1 signaling in normal mouse gastrocnemius muscle was sufficient to promote myofiber hypertrophy, which was accompanied by an increase in circulating and tissue-resident VEGF levels and high capillary vessel densities at focal regions of high Akt transgene expression. In a rabbit hind limb model of vascular insufficiency, intramuscular activation of Akt1 signaling promoted collateral and capillary vessel formation and an accompanying increase in limb perfusion. These data suggest that myogenic Akt signaling controls both fiber hypertrophy and angiogenic growth factor synthesis, illustrating a mechanism through which blood vessel recruitment can be coupled to normal tissue growth.

Reversal of GATA-6 Downregulation Promotes Smooth Muscle Differentiation and Inhibits Intimal Hyperplasia in Balloon-Injured Rat Carotid Artery

The GATA-6 transcription factor is expressed in quiescent vascular smooth muscle cells (VSMCs) in culture, and levels of its transcript are rapidly downregulated on mitogen stimulation. In this study, we demonstrate that the GATA-6 transcript, protein, and DNA-binding activity are downregulated in rat carotid arteries on balloon injury. Downregulation was detected at 1 and 3 days after injury and recovered by 7 days. To assess the role of GATA-6 downregulation in injury-induced vascular lesion formation, adenoviral vectors were used to express wild-type human GATA-6 cDNA (Ad-GATA6) or an inactive mutant cDNA that lacks a portion of the zinc-finger domain (Ad-GATA6DeltaZF). Adenovirus-mediated GATA-6 gene transfer to the vessel wall after balloon injury partially restored the levels of GATA-6 protein and DNA-binding activity to before injury levels. The local delivery of Ad-GATA6 but not Ad-GATA6DeltaZF inhibited lesion formation by 46% relative to saline control and 50% relative to a control adenovirus that expressed lacZ. Local delivery of Ad-GATA6 also reversed changes in the expression patterns of smooth muscle myosin heavy chain, smooth muscle alpha-actin, calponin, vinculin, metavinculin, and proliferating cell nuclear antigen that are associated with injury-induced VSMC phenotypic modulation. These data indicate that the injury-induced downregulation of GATA-6 is an essential feature of VSMC phenotypic modulation that contributes to vessel lesion formation.

Vascular Endothelial Growth Factor Attenuates Myocardial Ischemia-Reperfusion Injury

BACKGROUND Hypoxic endothelial cell activation plays a key role in the myocardial dysfunction resulting from ischemia-reperfusion injury. Recent evidence suggests that vascular endothelial growth factor (VEGF) may, in addition to promoting angiogenesis, modulate various aspects of endothelial function and repair. We examined whether administration of VEGF in the cardioplegic solution might have a beneficial effect on myocardial ischemia-reperfusion injury in an isolated rat heart model. METHODS Hearts from Sprague-Dawley rats were perfused with Krebs-Henseleit solution in a modified Langendorff apparatus. Percent recovery of cardiac output, coronary flow, stroke work, and percent increase in coronary vascular resistance were measured after 2 hours of global ischemia and 40 minutes of reperfusion. Coronary effluent was collected after ischemia and reperfusion for measurement of creatine kinase. RESULTS Hearts receiving cardioplegia solution containing 125 microg VEGF showed significantly improved recovery of cardiac output, coronary flow, and stroke work, and significantly reduced coronary vascular resistance compared with hearts receiving hyperkalemic cardioplegia only (p < 0.05). Coadministration of a nitric oxide synthase inhibitor attenuated the VEGF-induced cardiprotective effects. Hearts treated with VEGF released significantly less creatine kinase compared with control hearts. CONCLUSIONS Addition of VEGF to hyperkalemic cardioplegia protects against myocardial ischemia-reperfusion injury in the isolated rat heart.

Adenovirus-Mediated Delivery of Fas Ligand Inhibits Intimal Hyperplasia After Balloon Injury in Immunologically Primed Animals

BACKGROUND Adenoviral constructs have been used for studies of injury-induced vascular hyperplasia in immunologically naive laboratory animals, but their usefulness for intra-arterial gene therapy may be limited by the prevalence of preexisting immunity to adenovirus in the patient population. Here, we explored the efficacy of adenovirus-mediated transfer of Fas ligand, a cytotoxic gene with immunomodulatory properties, in inhibiting injury-induced vascular lesion formation in both naive and immunologically primed animals. METHODS AND RESULTS Lesion formation was evaluated in balloon-injured carotid arteries of naive and adenovirus-immunized rats that were infected with adenoviral constructs expressing Fas ligand (Ad-FasL), the cyclin-dependent kinase inhibitor p21 (Ad-p21), or beta-galactosidase (Ad-betagal). In naive rats, Ad-FasL induced apoptosis in medial vascular smooth muscle cells and inhibited intimal hyperplasia by 60% relative to Ad-betagal-treated vessels (P<0.05), whereas the cytostatic agent Ad-p21 decreased lesion size by 58% (P<0.05). In animals preimmunized with an adenoviral vector containing no transgene, Ad-FasL significantly inhibited neointima formation (73% reduction, P<0.05), but Ad-p21 failed to inhibit neointima formation relative to controls. Immunologically primed rats displayed robust T-cell infiltration in Ad-p21- and Ad-betagal-treated vessels, but T-cell infiltration was markedly attenuated in Ad-FasL-treated vessels. CONCLUSIONS Our data demonstrate that adenovirus-mediated Fas ligand delivery can inhibit intimal hyperplasia in both immunologically primed and naive animals, whereas the efficacy of an adenovirus-mediated p21 delivery is limited to immunologically naive animals. This study documents, for the first time, the therapeutic efficacy of intravascular adenoviral gene transfer in animals with preexisting immunity to adenovirus.

Regulation of smooth muscle cell migration and integrin expression by the Gax transcription factor

Homeobox transcription factors specify body plan by regulating differentiation, proliferation, and migration at a cellular level. The homeobox transcription factor Gax is expressed in quiescent vascular smooth muscle cells (VSMCs), and its expression is downregulated by vascular injury or other conditions that lead to VSMC proliferation. Previous investigations demonstrate that Gax may regulate VSMC proliferation by upregulating the cyclin-dependent kinase (cdk) inhibitor p21. Here we examined whether Gax influences VSMC migration, a key feature in the development of stenotic lesions after balloon injury. Transduction of a Gax cDNA inhibited the migratory response of VSMCs toward PDGF-BB, basic fibroblast growth factor, or hepatocyte growth factor/scatter factor. Gax expression also inhibited migration of NIH.3T3 fibroblasts and embryonic fibroblasts lacking p53. Gax was unable to inhibit the migration of fibroblasts lacking p21, but this effect could be restored in these cells by providing exogenous p21 or by overexpressing another cdk inhibitor, p16. Flow cytometric analysis implicated a Gax-mediated downregulation of alpha(v)beta(3) and alpha(v)beta(5) integrin expression in VSMCs as a potential cause for reduced cell motility. Gax specifically downregulated beta(3) and beta(5) in VSMCs in culture and after acute vascular injury in vivo. Repression of integrin expression was also found in NIH 3T3 cells and p53 knockout fibroblasts, but not in p21-knockout fibroblasts, unless these cells express exogenous p21 or p16. These data suggest that cycle progression, integrin expression, and cell migration can be regulated in VSMCs by the homeobox gene product Gax.

Akt signaling mediates VEGF/VPF vascular permeability in vivo

VEGF is an endothelial cell cytokine that promotes angiogenesis and enhances microvascular permeability. Recently, it has been shown that the protein kinase Akt functions in a key intercellular signaling pathway downstream of VEGF. Here, we employed adenovirus‐mediated gene transfer in conjunction with the Miles assay in hairless albino guinea pigs to assess the role of Akt signaling in vascular permeability. VEGF‐induced vascular permeability was blocked by the transduction of a dominant negative mutant of Akt. Conversely, transduction of a constitutively active form of Akt promoted vascular permeability in a manner similar to VEGF protein administration. This Akt‐mediated increase in vascular permeability was inhibited by the eNOS inhibitor L‐NAME. These data show that Akt signaling is both necessary and sufficient for vascular permeability in an in vivo model.

Fas Ligand Overexpression on Allograft Endothelium Inhibits Inflammatory Cell Infiltration and Transplant-Associated Intimal Hyperplasia

Despite recent advances in immunosuppressive therapy, accelerated coronary atherosclerosis remains a major problem in the long-term survival of transplant recipients. Chronic graft vasculopathy is believed to result from recipient inflammatory responses, and it is characterized by early mononuclear cell infiltration of the transplanted vessel. Here we show that endothelial cells can be genetically modified to overexpress functional, cell-surface Fas ligand (FasL) by adenovirus-mediated gene transfer without undergoing self-destruction. In a rodent model of transplant graft vasculopathy, endothelial overexpression of FasL attenuated T cell and macrophage infiltration at 1 wk posttransplantation. These vessels also displayed reduced neointima formation at one and 2 mo posttransplantation. These results indicate that inhibition of the early inflammatory response to allografted vessels by endothelial cell-specific overexpression of FasL may have utility in the treatment of transplant arteriosclerosis.