The p21 cyclin-dependent kinase inhibitor suppresses tumorigenicity in vivo.

The p21 gene encodes a cyclin-dependent kinase inhibitor that affects cell-cycle progression, but the potential of this gene product to serve as a tumour suppressor in vivo has not been established. In this report, we show that the growth of malignant cells in vitro and in vivo is inhibited by expression of p21. Expression of p21 resulted in an accumulation of cells in G0/G1, altered morphology, and cell differentiation, but apoptosis was not induced. Introduction of p21 with adenoviral vectors into malignant cells completely suppressed their growth in vivo and also reduced the growth of established pre-existing tumours. Gene transfer of p21 may provide a molecular genetic approach to arresting cancer cell growth by committing malignant cells irreversibly to a pathway of terminal differentiation.

Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury.

Here we defined the main viral determinant of Ebola virus pathogenicity; synthesis of the virion glycoprotein (GP) of Ebola virus Zaire induced cytotoxic effects in human endothelial cells in vitro and in vivo. This effect mapped to a serine-threonine-rich, mucin-like domain of this type I transmembrane glycoprotein, one of seven gene products of the virus. Gene transfer of GP into explanted human or porcine blood vessels caused massive endothelial cell loss within 48 hours that led to a substantial increase in vascular permeability. Deletion of the mucin-like region of GP abolished these effects without affecting protein expression or function. GP derived from the Reston strain of virus, which causes disease in nonhuman primates but not in man, did not disrupt the vasculature of human blood vessels. In contrast, the Zaire GP induced endothelial cell disruption and cytotoxicity in both nonhuman primate and human blood vessels, and the mucin domain was required for this effect. These findings indicate that GP, through its mucin domain, is the viral determinant of Ebola pathogenicity and likely contributes to hemorrhage during infection.

Immunization for Ebola virus infection.

Infection by Ebola virus causes rapidly progressive, often fatal, symptoms of fever, hemorrhage and hypotension. Previous attempts to elicit protective immunity for this disease have not met with success. We report here that protection against the lethal effects of Ebola virus can be achieved in an animal model by immunizing with plasmids encoding viral proteins. We analyzed immune responses to the viral nucleoprotein (NP) and the secreted or transmembrane forms of the glycoprotein (sGP or GP) and their ability to protect against infection in a guinea pig infection model analogous to the human disease. Protection was achieved and correlated with antibody titer and antigen-specific T-cell responses to sGP or GP. Immunity to Ebola virus can therefore be developed through genetic vaccination and may facilitate efforts to limit the spread of this disease.

Heme oxygenase-1 protects against vascular constriction and proliferation.

Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.

Site-specific gene expression in vivo by direct gene transfer into the arterial wall.

A recombinant beta-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.

Recombinant gene expression in vivo within endothelial cells of the arterial wall.

A technique for the transfer of endothelial cells and expression of recombinant genes in vivo could allow the introduction of proteins of therapeutic value in the management of cardiovascular diseases. Porcine endothelial cells expressing recombinant beta-galactosidase from a murine amphotropic retroviral vector were introduced with a catheter into denuded iliofemoral arteries of syngeneic animals. Arterial segments explanted 2 to 4 weeks later contained endothelial cells expressing beta-galactosidase, an indication that they were successfully implanted on the vessel wall.

Gene therapy for vascular smooth muscle cell proliferation after arterial injury.

Accumulation of vascular smooth muscle cells as a consequence of arterial injury is a major feature of vascular proliferative disorders. Molecular approaches to the inhibition of smooth muscle cell proliferation in these settings could potentially limit intimal expansion. This problem was approached by introducing adenoviral vectors encoding the herpesvirus thymidine kinase (tk) into porcine arteries that had been injured by a balloon on a catheter. These smooth muscle cells were shown to be infectable with adenoviral vectors, and introduction of the tk gene rendered them sensitive to the nucleoside analog ganciclovir. When this vector was introduced into porcine arteries immediately after a balloon injury, intimal hyperplasia decreased after a course of ganciclovir treatment. No major local or systemic toxicities were observed. These data suggest that transient expression of an enzyme that catalyzes the formation of a cytotoxic drug locally may limit smooth muscle cell proliferation in response to balloon injury.

Distinct cellular interactions of secreted and transmembrane Ebola virus glycoproteins.

The mechanisms by which Ebola virus evades detection and infects cells to cause hemorrhagic fever have not been defined, though its glycoprotein, synthesized in either a secreted or transmembrane form, is likely involved. Here the secreted glycoprotein was found to interact with neutrophils through CD16b, the neutrophil-specific form of the Fc gamma receptor III, whereas the transmembrane glycoprotein was found to interact with endothelial cells but not neutrophils. A murine retroviral vector pseudotyped with the transmembrane glycoprotein preferentially infected endothelial cells. Thus, the secreted glycoprotein inhibits early neutrophil activation, which likely affects the host response to infection, whereas binding of the transmembrane glycoprotein to endothelial cells may contribute to the hemorrhagic symptoms of this disease.

Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product.

Vascular smooth muscle cell (SMC) proliferation in response to injury is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. The retinoblastoma gene product (Rb) is present in the unphosphorylated and active form in quiescent primary arterial SMCs, but is rapidly inactivated by phosphorylation in response to growth factor stimulation in vitro. A replication-defective adenovirus encoding a nonphosphorylatable, constitutively active form of Rb was constructed. Infection of cultured primary rat aortic SMCs with this virus inhibited growth factor-stimulated cell proliferation in vitro. Localized arterial infection with the virus at the time of balloon angioplasty significantly reduced SMC proliferation and neointima formation in both the rat carotid and porcine femoral artery models of restenosis. These results demonstrate the role of Rb in regulating vascular SMC proliferation and suggest a gene therapy approach for vascular proliferative disorders associated with arterial injury.

Recombinant platelet-derived growth factor B gene expression in porcine arteries induce intimal hyperplasia in vivo.

Platelet-derived growth factor (PDGF) B chain induces cell proliferation in vitro and is associated with arterial lesions that cause cardiovascular disease. However, it has been difficult to document the biological response to PDGF B gene expression in arteries in vivo. To determine the biologic effects of this growth factor in vivo, we have introduced an eukaryotic expression vector plasmid encoding recombinant PDGF B by direct gene transfer into porcine iliofemoral arteries using DNA liposome complexes. The presence of PDGF B plasmid DNA and expression of recombinant mRNA were confirmed by polymerase chain reaction analysis, and recombinant PDGF protein was demonstrated by immunohistochemistry. Intimal thickening was observed in porcine arteries 21 days following transfection with the recombinant PDGF B gene compared with arteries transduced with a control gene, E. coli beta-galactosidase. An eightfold increase in intimal to medial ratio was present in PDGF B gene transfected arteries compared with control transfected arteries (P = 0.001). This study suggests that expression of a recombinant PDGF B gene in vivo can play a role in the induction of intimal hyperplasia, which can lead to cardiovascular diseases.

Regulation of cellular proliferation and intimal formation following balloon injury in atherosclerotic rabbit arteries.

Injury to atherosclerotic arteries induces the expression of growth regulatory genes that stimulate cellular proliferation and intimal formation. Intimal expansion has been reduced in vivo in nonatherosclerotic balloon-injured arteries by transfer of genes that inhibit cell proliferation. It is not known, however, whether vascular cell proliferation can be inhibited after injury in more extensively diseased atherosclerotic arteries. Accordingly, the purpose of this study was to investigate whether expression of recombinant genes in atherosclerotic arteries after balloon injury could inhibit intimal cell proliferation. To test this hypothesis, we examined the response to balloon injury in atherosclerotic rabbit arteries after gene transfer of herpesvirus thymidine kinase gene (tk) and administration of ganciclovir. Smooth muscle cells from hyperlipidemic rabbit arteries infected with adenoviral vectors encoding tk were sensitive to ganciclovir, and bystander killing was observed in vitro. In atherosclerotic arteries, a human placental alkaline phosphatase reporter gene was expressed in intimal and medial smooth muscle cells and macrophages, identifying these cells as targets for gene transfer. Expression of tk in balloon-injured hyperlipidemic rabbit arteries followed by ganciclovir treatment resulted in a 64% reduction in intimal cell proliferation 7 d after gene transfer (P = 0.004), and a 35-49% reduction in internal area 21 d after gene transfer, compared with five different control groups (P < 0.05). Replication of smooth muscle cells and macrophages was inhibited by tk expression and ganciclovir treatment. These findings indicate that transfer of a gene that inhibits cellular proliferation limits the intimal area in balloon-injured atherosclerotic arteries. Molecular approaches to the inhibition of cell proliferation in atherosclerotic arteries constitute a possible treatment for vascular proliferative diseases.

Fibroblast growth factor stimulates angiotensin converting enzyme expression in vascular smooth muscle cells. Possible mediator of the response to vascular injury.

Angiotensin converting enzyme (ACE) activity contributes to the vascular response to injury because ACE inhibition limits neointima formation in rat carotid arteries after balloon injury. To investigate the mechanisms by which ACE may contribute to vascular smooth muscle cell (VSMC) proliferation, we studied expression of ACE in vivo after injury and in vitro after growth factor stimulation. ACE activity 14 d after injury was increased 3.6-fold in the injured vessel. ACE expression, measured by immunohistochemistry, became apparent at 7 d in the neointima and at 14 d was primarily in the most luminal neointimal cells. To characterize hormones that induce ACE in vivo, cultured VSMC were exposed to steroids and growth factors. Among steroids, only glucocorticoids stimulated ACE expression with an 8.0 +/- 2.1-fold increase in activity and a 6.5-fold increase in mRNA (30 nM dexamethasone for 72 h). Among growth factors tested, only fibroblast growth factor (FGF) stimulated ACE expression (4.2 +/- 0.7-fold increase in activity and 1.6-fold increase in mRNA in response to 10 ng/ml FGF for 24 h). Dexamethasone and FGF were synergistic at the indicated concentrations inducing 50.6 +/- 12.4-fold and 32.5-fold increases in activity and mRNA expression, respectively. In addition, when porcine iliac arteries were transfected with recombinant FGF-1 (in the absence of injury), ACE expression increased in neointimal VSMC, to the same extent as injured, nontransfected arteries. The data suggest a temporal sequence for the response to injury in which FGF induces ACE, ACE generates angiotensin II, and angiotensin II stimulates VSMC growth in concert with FGF.

Responses of coronary arteries of cardiac transplant patients to acetylcholine.

Accelerated coronary atherosclerosis is a major cause of graft failure after heart transplantation. Graft atherosclerosis is typically diffuse and difficult to detect even with coronary arteriography. Recently, acetylcholine was shown to dilate blood vessels by releasing a vasorelaxant substance from the endothelium (endothelium-derived relaxing factor). We have demonstrated paradoxical vasoconstriction induced by acetylcholine both early and late in the course of coronary atherosclerosis in patients, suggesting an association of endothelial dysfunction and atherosclerosis. In this report, we tested the hypothesis that coronary arteries of heart transplant patients can show endothelial dysfunction before or in the early stages of angiographically evident coronary atherosclerosis. Acetylcholine was infused into the left anterior descending artery of 13 heart transplant patients at 12 (n = 9) and 24 (n = 4) mo after transplantation. Vascular responses were evaluated by quantitative angiography. Among patients with angiographically smooth coronary arteries, relatively few (6/25) arterial segments had preserved vasodilator responses, while the majority failed to dilate (10/25) or paradoxically constricted (9/25). Angiographically irregular coronary arteries were present in three patients, in whom 8/10 segments showed marked paradoxical constriction and the remaining 2/10 failed to dilate. Only 1 of 13 patients retained appropriate dilation to acetylcholine in all segments. Nitroglycerin, which acts directly on vascular smooth muscle, dilated nearly all segments. No clinical features of the patients, including myocardial rejection appeared to correlate with the impaired functional response of vessels. Thus impaired response to acetylcholine is a common early finding in heart transplant patients and emphasizes the potential importance of endothelial dysfunction in the development of atherosclerosis.

Atherosclerosis influences the vasomotor response of epicardial coronary arteries to exercise.

We studied the vasomotion of epicardial coronary arteries during exercise and tested the hypotheses that abnormal vasoconstriction is related to the presence of atherosclerosis and may be related to endothelial dilator dysfunction. During cardiac catheterization quantitative coronary angiography was performed in 21 patients during supine bicycle exercise. 21 of 28 smooth, angiographically normal vessel segments dilated (14.0 +/- 1.8%) during exercise; four smooth segments did not change whereas only three constricted. In contrast, 15 of 16 vessel segments with irregularities constricted in response to exercise (17.0 +/- 0.1%) with only one segment dilating. All 10 stenotic segments constricted to exercise (23 +/- 4%). Six patients also received intracoronary acetylcholine before exercise to test endothelium-dependent dilator function. In five of six patients all nine vessel segments showed the same directional response to acetylcholine and exercise. Three irregular and two stenotic segments constricted with acetylcholine (51 +/- 21%) and exercise (9.0 +/- 0.6%). In contrast, four smooth segments dilated to acetylcholine (19 +/- 6%) and exercise (9 +/- 1%). Both exercise and acetylcholine generally dilated smooth but constricted irregular and stenosed coronary segments. It appears likely that atherosclerosis plays an important role in the abnormal vasomotion of diseased coronary arteries during exercise and the pattern of abnormality suggests impairment of vasodilator function.

Transcriptional regulation of NF-kappa B2: evidence for kappa B-mediated positive and negative autoregulation.

NF-kappa B is an inducible transcription factor complex which regulates the expression of a variety of genes which are involved in the immune, inflammatory, and acute-phase responses. The maintenance of NF-kappa B activity in stimulated cells requires ongoing protein synthesis, suggesting several modes of regulation. In this report, we have characterized the transcriptional regulation of one family member, NF-kappa B2. The genomic structure and sequence of NF-kappa B2 revealed the presence of two promoters and at least four kappa B regulatory elements, which mediate responsiveness to phorbol myristate acetate and tumor necrosis factor alpha. Similar to other NF-kappa B family members, NF-kappa B2 is positively autoregulated. In contrast to other family members, we find that kappa B elements in the NFKB2 promoter can also mediate transcriptional repression in the absence of NF-kappa B. We identified a nuclear complex which binds specifically to a subset of kappa B-related sites but not to the canonical kappa B element. Because of its putative inhibitory or repressive effect, this binding activity has been termed Rep-kappa B. This mechanism of repressing basal NF-kappa B2 transcription in an inactivated state enables the cell to tightly control NF-kappa B2 activity. These data demonstrate that a novel mode of kappa B-dependent regulation is mediated by specific kappa B sites in the NFKB2 promoter.

Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-kappa B subunits in endothelial and epithelial cells.

Vascular cell adhesion molecule 1 (VCAM-1) is expressed in both endothelial and epithelial cell types, where it contributes to lymphocyte migration to sites of inflammation. Its expression is regulated by cytokines, in part through two kappa B-like regulatory elements. Because NF-kappa B can be composed of multiple alternative subunits with differential effects on gene expression, the role of different specific NF-kappa B family members subunits in VCAM-1 regulation is unknown. In this report, we define the contribution of different NF-kappa B family members to VCAM-1 gene regulation. We show that both kappa B sites in the VCAM-1 enhancer are required to optimally stimulate gene expression, but the enhancer is differentially regulated by specific combinations of NF-kappa B subunits. At low concentrations, RelA(p65) acted in concert with the approximately 50-kDa product of p105 NF-kappa B, NF-kappa B1(p50), to stimulate transcription, and at high concentrations, RelA(p65) alone stimulated the VCAM-1 promoter. In contrast, NF-kappa B2 inhibited functional activation of the VCAM reporter by p65. Consistent with this finding, an additional binding complex was detected by using recombinant NF-kappa B2(p49)/RelA(p65) with radiolabeled VCAM kappa B site probes. Interestingly, the human immunodeficiency virus enhancer responded differently to stimulation by NF-kappa B subunits, with optimal response to p49(100)/p65. Analysis of NF-kappa B mRNA in human umbilical vein endothelial cells revealed that nfkb1, nfkb2, and relA NF-kappa B but not c-rel were induced by tumor necrosis factor alpha and lipopolysaccharide, which also induce VCAM-1. These data suggest that specific subunits of NF-kappa B regulate VCAM-1 and differentially activate other genes in these cells.

Adenovirus-mediated gene transfer of VEGF(121) improves lower-extremity endothelial function and flow reserve.

BACKGROUND: Vascular endothelial growth factor (VEGF) currently is being evaluated in clinical angiogenesis trials involving patients with peripheral arterial disease. We hypothesized that delivery of VEGF to the skeletal muscle of the lower extremity using an adenoviral vector (Ad(GV)VEGF(121.10)) would improve peripheral endothelial function. Accordingly, we investigated lower-extremity endothelial function in patients enrolled in a Phase I adenovirus-mediated gene delivery trial of VEGF(121.10). METHODS AND RESULTS: Blood flow to the index extremity was measured by thermodilution at baseline and 30 days after administration of Ad(GV)VEGF(121.10), in response to the infusion of endothelium-dependent and -independent agonists (acetylcholine and nitroglycerin, respectively) into the ipsilateral femoral artery. There was no difference in basal flow before or after treatment with Ad(GV)VEGF(121.10). In response to acetylcholine (150 microg/min and 300 microg/min), there was a 0.9-fold (0.33+/-0.03 to 0.32+/-0.03 L/min) and 1.2-fold (0.33+/-0.03 to 0.490+/-0.02 L/min) change in flow before Ad(GV)VEGF(121.10) treatment. After Ad(GV)VEGF(121.10) treatment, flow increased 2.4-fold (0.310+/-0.04 to 0.730+/-0.10 L/min) and 2.3-fold (0.31+/-0.04 to 0.7+/-0.08 L/min), respectively (P<0.05 before Ad(GV)VEGF(121.10) treatment versus after Ad(GV)VEGF(121.10) for both doses). Infusion of nitroglycerin resulted in a 1.8-fold increase in flow before Ad(GV)VEGF(121.10) (0.33+/-0.03 to 0.58+/-0.06 L/min) compared with a 2.4-fold increase (0.31+/-0.04 to 0.73+/-0.09 L/min) after Ad(GV)VEGF(121.10) (P=NS before Ad(GV)VEGF(121.10) versus after Ad(GV)VEGF(121.10)). Lower-extremity flow reserve increased in all patients in response to at least 1 dose of acetylcholine. Peak walking times increased concomitant with improvement in endothelial function. CONCLUSIONS: Adenoviral gene transfer of VEGF(121.10) appears to modulate endothelial function and lower-extremity flow reserve in patients with peripheral arterial disease.

Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation.

BACKGROUND: We examined the effect of NO on the proliferation and cell cycle regulation of human aortic vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: The NO donor diethylenetriamineNONOate (10(-5) to 10(-3) mol/L) inhibited proliferation in response to 10% fetal calf serum (FCS) and 100 ng/mL platelet-derived growth factor-BB in a concentration-dependent manner. This effect was not observed with disintegrated diethylenetriamineNONOate or with the parent compound, diethylenetriamine. Adenoviral transfection of endothelial NO synthase (NOS) inhibited proliferation in response to FCS, which was prevented with N(G)-nitro-L-arginine methyl ester. NOS overexpression did not inhibit proliferation in response to platelet-derived growth factor, although the transfection efficiency and protein expression were similar to those of FCS-stimulated cells. Nitrate release was selectively enhanced from FCS-treated cells, indicating that NOS was activated by FCS only. NO caused G(1) cell cycle arrest. Cytotoxicity was determined with trypan blue exclusion, and apoptosis was assessed with DNA fragmentation. Cyclin-dependent kinase 2 expression level, threonine phosphorylation, and kinase activity were inhibited. Cyclin A expression was blunted, whereas cyclin E remained unchanged. p21 expression was induced, and p27 remained unaltered. The effect on cyclin A and p21 started within 6 hours and preceded the changes in cell cycle distribution. Proliferation in response to 10% FCS was barely inhibited with 8-bromo-cGMP (10(-3) mol/L) but was blunted with both forskolin and 8-bromo-cAMP. Proliferation in response to 2% FCS was inhibited with 8-bromo-cGMP, but it did not mimic the cell cycle effects of NO. CONCLUSIONS: NO inhibits VSMC proliferation by specifically changing the expression and activity of cell cycle regulatory proteins, which may occur independent of cGMP. Adenoviral overexpression of endothelial NOS represents a cytostatic strategy for gene therapy of vascular disease.

Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation.

BACKGROUND: The cyclin-dependent kinase inhibitors (CKIs) have different patterns of expression in vascular diseases. The Kip/Cip CKIs, p27(Kip1) and p21(Cip1), are upregulated during arterial repair and negatively regulate the growth of vascular smooth muscle cells (VSMCs). In contrast, the Ink CKI, p16(Ink4), is not expressed in vascular lesions. We hypothesized that a variation in the inactivation of cdk2 and cdk4 during the G(1) phase of the cell cycle by p27(Kip1), p21(Cip1), and p16(Ink4) leads to different effects on VSMC growth in vitro and in vivo. METHODS AND RESULTS: The expression of p27(Kip1) and p21(Cip1) in serum-stimulated VSMCs inactivated cdk2 and cdk4, leading to G(1) growth arrest. p16(Ink4) inhibited cdk4, but not cdk2, kinase activity, producing partial inhibition of VSMC growth in vitro. In an in vivo model of vascular injury, overexpression of p27(Kip1) reduced intimal VSMC proliferation by 52% (P<0.01) and the intima/media area ratio by 51% (P<0.005) after vascular injury and gene transfer to pig arteries, when compared with control arteries. p16(Ink4) was a weak inhibitor of intimal VSMC proliferation in injured arteries (P=NS), and it did not significantly reduce intima/media area ratios (P=NS), which is consistent with its minor effects on VSMC growth in vitro. CONCLUSIONS: p27(Kip1) and p21(Cip1) are potent inhibitors of VSMC growth compared with p16(Ink4) because of their different molecular mechanisms of cyclin-dependent kinase inhibition in the G(1) phase of the cell cycle. These findings have important implications for our understanding of the pathophysiology of vascular proliferative diseases and for the development of molecular therapies.