Hong San

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.

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.

Differential Effects of the Cyclin-Dependent Kinase Inhibitors p27Kip1, p21Cip1, and p16Ink4 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.

A farnesyltransferase inhibitor prevents both the onset and late progression of cardiovascular disease in a progeria mouse model

Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic form of human premature aging. Death occurs at a mean age of 13 years, usually from heart attack or stroke. Almost all cases of HGPS are caused by a de novo point mutation in the lamin A (LMNA) gene that results in production of a mutant lamin A protein termed progerin. This protein is permanently modified by a lipid farnesyl group, and acts as a dominant negative, disrupting nuclear structure. Treatment with farnesyltransferase inhibitors (FTIs) has been shown to prevent and even reverse this nuclear abnormality in cultured HGPS fibroblasts. We have previously created a mouse model of HGPS that shows progressive loss of vascular smooth muscle cells in the media of the large arteries, in a pattern that is strikingly similar to the cardiovascular disease seen in patients with HGPS. Here we show that the dose-dependent administration of the FTI tipifarnib (R115777, Zarnestra) to this HGPS mouse model can significantly prevent both the onset of the cardiovascular phenotype as well as the late progression of existing cardiovascular disease. These observations provide encouraging evidence for the current clinical trial of FTIs for this rare and devastating disease.

Detection of atherosclerosis using a novel positron-sensitive probe and 18-fluorodeoxyglucose (FDG).

Inflammation contributes to atherosclerotic plaque remodeling, enlargement and rupture. Non-invasive imaging of coronary artery inflammation could help target therapy to ‘vulnerable’ atheromata, but is limited because of small tissue mass and arterial motion. Local radiopharmaceutical imaging may overcome some of these limitations. We used a positron-sensitive fiberoptic probe, which can distinguish positron emissions from annihilation photons, to identify diseased from healthy endothelium in an atherosclerotic model. New Zealand White rabbits underwent Fogarty-catheter injury of an iliac artery and then were fed a high-fat diet for 3 weeks. Fasted animals received 90-180 MBq of 18-fluorodeoxyglucose (FDG) 2-4 h before sacrifice and harvest of injured and uninjured iliacs. Arteries were incised longitudinally and the probe was placed in contact with the arterial intima. Multiple measurements were obtained along 1 cm artery segments in 60 s intervals, and corrected for 18F decay and background. Measurements were recorded over 93 injured and normal artery segments in 11 animals. Mean probe Z-scores were 4.8-fold higher (CI 3.4-6.3) over injury atherosclerosis compared with uninjured normal iliac artery segments (P<0.001). Gamma counting confirmed that injured artery segments accumulated more FDG per gram than did normal segments (0.203%·kg injected dose per gram of tissue versus 0.042, P<0.001). Non-arterial tissue also accumulated FDG avidly, particularly reticuloendothelial tissues and blood. Delayed sacrifice, 4 h compared with 2 h after animal FDG injection, further reduced blood background counts and improved the signal-to-noise ratio. Histopathology confirmed that injured iliac artery had significantly higher intimal and medial cross-sectional area compared with uninjured artery. Injured artery also had significantly higher macrophage and smooth muscle cell density. Positron-sensitive probe counts correlated with the intima to media ratio (r = 0.63, P = 0.03). Our positron-sensitive probe distinguishes atherosclerotic from healthy artery in a blood-free field. Intravascular study of plaque biology may be feasible using FDG and a positron-sensitive probe.

TGF-β Signaling Mediates Endothelial-to-Mesenchymal Transition (EndMT) During Vein Graft Remodeling

In vivo endothelial cell fate mapping demonstrates that TGF-β signaling is a central pathway regulating the endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling. Negative Remodeling In coronary bypass surgery, veins are grafted to arteries, in hopes of generating a functional vessel. Although a routine procedure, grafting can result in a negative remodeling process—with a poorly understood underlying mechanism. Here, Cooley and colleagues linked vein graft stenosis (blood vessel narrowing) and negative remodeling to a process called the endothelial-to-mesenchymal transition (EndMT). Although well known during development, the presence of EndMT in the vasculature is less documented and therefore represents a possible new target in preventing graft failure. The authors tracked endothelial cells in mice using yellow fluorescent protein (YFP), and saw that these cells lining the vessel walls contributed to arterial thickening (neointima formation) after vein grafting by first converting to mesenchymal cells. EndMT occurred via transforming growth factor–β (TGF-β) signaling, specifically through intermediates Smad2/3 and Slug. Knowing the pathway at play is important for translation to the clinic because therapeutics can be designed to target these signaling molecules. Indeed, the authors found that blocking TGF-β with an antibody or knocking down Smad2 in vivo in mice prevented EndMT. The mesenchymal transition was also noted in failed vein grafts taken from patients, suggesting that EndMT is also present in humans and contributes to graft failure and restenosis. More testing is required in human samples to confirm the mouse data, but EndMT appears to be a viable target for improving graft outcomes after surgery in patients. Veins grafted into an arterial environment undergo a complex vascular remodeling process. Pathologic vascular remodeling often results in stenosed or occluded conduit grafts. Understanding this complex process is important for improving the outcome of patients with coronary and peripheral artery disease undergoing surgical revascularization. Using in vivo murine cell lineage–tracing models, we show that endothelial-derived cells contribute to neointimal formation through endothelial-to-mesenchymal transition (EndMT), which is dependent on early activation of the Smad2/3-Slug signaling pathway. Antagonism of transforming growth factor–β (TGF-β) signaling by TGF-β neutralizing antibody, short hairpin RNA–mediated Smad3 or Smad2 knockdown, Smad3 haploinsufficiency, or endothelial cell–specific Smad2 deletion resulted in decreased EndMT and less neointimal formation compared to controls. Histological examination of postmortem human vein graft tissue corroborated the changes observed in our mouse vein graft model, suggesting that EndMT is operative during human vein graft remodeling. These data establish that EndMT is an important mechanism underlying neointimal formation in interpositional vein grafts, and identifies the TGF-β–Smad2/3–Slug signaling pathway as a potential therapeutic target to prevent clinical vein graft stenosis.

Heme Oxygenase-1 Deficiency Accelerates Formation of Arterial Thrombosis Through Oxidative Damage to the Endothelium, Which Is Rescued by Inhaled Carbon Monoxide

Heme oxygenase (HO)-1 (encoded by Hmox1) catalyzes the oxidative degradation of heme to biliverdin and carbon monoxide. HO-1 is induced during inflammation and oxidative stress to protect tissues from oxidative damage. Because intravascular thrombosis forms at sites of tissue inflammation, we hypothesized that HO-1 protects against arterial thrombosis during oxidant stress. To investigate the direct function of HO-1 on thrombosis, we used photochemical-induced vascular injury in Hmox1−/− and Hmox1+/+ mice. Hmox1−/− mice developed accelerated, occlusive arterial thrombus compared with Hmox1+/+ mice, and we detected several mechanisms accounting for this antithrombotic effect. First, endothelial cells in Hmox1−/− arteries were more susceptible to apoptosis and denudation, leading to platelet-rich microthrombi in the subendothelium. Second, tissue factor, von Willebrand Factor, and reactive oxygen species were significantly elevated in Hmox1−/− mice, consistent with endothelial cell damage and loss. Third, following transplantation of Hmox1−/− donor bone marrow into Hmox1+/+ recipients and subsequent vascular injury, we observed rapid arterial thrombosis compared with Hmox1+/+ mice receiving Hmox1+/+ bone marrow. Fourth, inhaled carbon monoxide and biliverdin administration rescued the prothrombotic phenotype in Hmox1−/− mice. Fifth, using a transcriptional analysis of arterial tissue, we found that HO-1 determined a transcriptional response to injury, with specific effects on cell cycle regulation, coagulation, thrombosis, and redox homeostasis. These data provide direct genetic evidence for a protective role of HO-1 against thrombosis and reactive oxygen species during vascular damage. Induction of HO-1 may be beneficial in the prevention of thrombosis associated with vascular oxidant stress and inflammation.

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.

Stat3-dependent acute Rantes production in vascular smooth muscle cells modulates inflammation following arterial injury in mice

Inflammation is a key component of arterial injury, with VSMC proliferation and neointimal formation serving as the final outcomes of this process. However, the acute events transpiring immediately after arterial injury that establish the blueprint for this inflammatory program are largely unknown. We therefore studied these events in mice and found that immediately following arterial injury, medial VSMCs upregulated Rantes in an acute manner dependent on Stat3 and NF-kappaB (p65 subunit). This led to early T cell and macrophage recruitment, processes also under the regulation of the cyclin-dependent kinase inhibitor p21Cip1. Unique to VSMCs, Rantes production was initiated by Tnf-alpha, but not by Il-6/gp130. This Rantes production was dependent on the binding of a p65/Stat3 complex to NF-kappaB-binding sites within the Rantes promoter, with shRNA knockdown of either Stat3 or p65 markedly attenuating Rantes production. In vivo, acute NF-kappaB and Stat3 activation in medial VSMCs was identified, with acute Rantes production after injury substantially reduced in Tnfa-/- mice compared with controls. Finally, we generated mice with SMC-specific conditional Stat3 deficiency and confirmed the Stat3 dependence of acute Rantes production by VSMCs. Together, these observations unify inflammatory events after vascular injury, demonstrating that VSMCs orchestrate the arterial inflammatory response program via acute Rantes production and subsequent inflammatory cell recruitment.

Catheter-mediated pulmonary vascular gene transfer and expression.

The study and treatment of pulmonary diseases may be greatly facilitated by in vivo expression of specific recombinant genes in the pulmonary vasculature and lung parenchyma. To evaluate the feasibility of gene transfer to the pulmonary vasculature, cationic liposomes and adenoviral vectors encoding a human placental alkaline phosphatase (hpAP) gene were delivered into a pulmonary artery of 24 pigs by percutaneous right heart catheterization. Pulmonary tissue was harvested within 20 minutes or 5, 14, or 28 days later and was analyzed for gene transfer and expression. Five days after exposure to liposomes or adenoviral vectors, transfer of DNA and expression of mRNA were demonstrated in transfected lung tissue. Recombinant alkaline phosphatase protein was observed in both the vasculature and in alveolar septa but not in the bronchi. Expression of hpAP protein was observed at 5 days, was diminished at 14 days, and was absent 28 days after gene transfer with both liposome and adenoviral vectors. No major adverse effects of gene expression were detected by histological examination of the transfected lung segments compared with control segments. Gene transfer to the lung by either vector was not associated with significant biochemical abnormalities or histological changes 5, 14, or 28 days later in other organs, including carotid artery, heart, liver, spleen, kidney, skeletal muscle, ovary, and testes. These studies demonstrate that after intravascular gene delivery to the lung, recombinant genes are expressed in the vasculature and alveoli. This approach may provide a useful model for the experimental study of pulmonary vascular diseases, including pulmonary fibrosis and pulmonary thrombosis disorders.