Nagadhara Dronadula

Overexpression of Urokinase by Plaque Macrophages Causes Histological Features of Plaque Rupture and Increases Vascular Matrix Metalloproteinase Activity in Aged Apolipoprotein E–Null Mice

Background— The mechanisms of atherosclerotic plaque rupture are poorly understood. Urokinase-type plasminogen activator (uPA) is expressed at elevated levels by macrophages in advanced human plaques. Patients with evidence of increased plasminogen activation have an elevated risk of major cardiovascular events. We used atherosclerotic mice to test the hypothesis that increased macrophage uPA expression in advanced plaques would cause histological features similar to those in ruptured human plaques. Methods and Results— Bone marrow from transgenic mice with increased macrophage uPA expression or nontransgenic controls (all apolipoprotein E-null [Apoe−/−]) was transplanted into 35-week-old Apoe−/− recipients, and innominate lesions and aortas were examined 8 to 13 weeks later. Donor macrophages accumulated in innominate lesions adjacent to plaque caps and in aortas, increasing uPA expression at both sites. Recipients of uPA-overexpressing macrophages had an increased prevalence of intraplaque hemorrhage (61% versus 13%; P=0.002) as well as increased lesion fibrin staining and fibrous cap disruption (P=0.06 for both). Transplantation of uPA-overexpressing macrophages increased aortic matrix metalloproteinase activity (40%; P=0.02). This increase was independent of matrix metalloproteinase-9. Conclusions— In advanced plaques of Apoe−/− mice, macrophage uPA overexpression causes intraplaque hemorrhage and fibrous cap disruption, features associated with human plaque rupture. uPA overexpression also increases vascular matrix metalloproteinase activity. These data provide a mechanism that connects macrophage uPA expression, matrix metalloproteinase activity, and plaque rupture features in mice. The data also suggest that elevated plaque plasminogen activator expression and plasminogen activation in humans may be causally linked to plaque rupture and cardiovascular events.

Transforming Growth Factor-β Signaling in Myogenic Cells Regulates Vascular Morphogenesis, Differentiation, and Matrix Synthesis

Objective— Transforming growth factor-&bgr; (TGF-&bgr;) signaling is required for normal vascular development. We aimed to discover the role of TGF-&bgr; signaling in embryonic smooth muscle cells (SMCs). Methods and Results— We bred mice with smooth muscle (SM) 22&agr;-Cre and Tgfbr2flox alleles to generate embryos in which the type II TGF-&bgr; receptor (TGFBR2; required for TGF-&bgr; signaling) was deleted in SMCs. Embryos were harvested between embryonic day (E) 9.5 and E18.5 and examined grossly, microscopically, and by histochemical and RNA analyses. SM22&agr;-Cre+/0 Tgfbr2flox/flox (knockout [KO]) embryos died before E15.5 with defects that included cardiac outflow tract abnormalities, persistence of the right dorsal aorta, and dilation of the distal aorta. Histological analyses suggested normal expression of SMC differentiation markers in KO aortas; however, RNA analyses showed that SMC differentiation markers were increased in KO cardiac outflow vessels but decreased in the descending aorta. KO aortas had only rare mature elastin deposits and contained abnormal aggregates of extracellular matrix proteins. Expression of several matrix proteins was significantly decreased in KO descending aortas but not in cardiac outflow vessels. Conclusion— TGF-&bgr; signaling in SMCs controls differentiation, matrix synthesis, and vascular morphogenesis. Effects of TGF-&bgr; on SMC gene expression appear to differ depending on the location of SMCs in the aorta.

Helper-Dependent Adenoviral Vector Achieves Prolonged, Stable Expression of Interleukin-10 in Rabbit Carotid Arteries but Does Not Limit Early Atherogenesis

Vascular gene therapy could potentially complement or replace current therapies for human atherosclerosis, while avoiding their side effects. However, development of vascular gene therapy is limited by lack of a useful vector. Helper-dependent adenovirus (HDAd) shows promise to overcome this barrier because, unlike first-generation adenovirus, HDAd achieves durable transgene expression in the artery wall with minimal inflammation. To begin to test whether HDAd, delivered to the artery wall, can limit atherosclerosis we constructed HDAd that expresses rabbit interleukin (IL)-10, a potent atheroprotective cytokine, and tested its activity in a rabbit model of early carotid atherogenesis. HDAd expressed immunoreactive, active IL-10 in vitro. In contrast to other HDAd-expressed transgenes, IL-10 expression from HDAd increased significantly between 3 days and 2 weeks after infusion and remained stable for at least 8 weeks. Rising, persistent IL-10 expression was associated with relative persistence of HDAdIL-10 genomes 4 weeks after infusion, compared with HDAdNull genomes. Surprisingly, IL-10 expression had no significant effects on atherosclerotic lesion size, macrophage content, or expression of either adhesion molecules or atherogenic cytokines. These results might be due to inadequate protein expression in vivo or lack of suitability of this rabbit model to reveal IL-10 therapeutic effects. IL-10 remains a promising agent for vascular gene therapy and HDAd remains a promising vector; however, proof of efficacy of HDAdIL-10 is elusive. Future preclinical studies will be aimed at increasing IL-10 expression levels and improving the sensitivity of this animal model to detect atheroprotective effects.

Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels

The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here, we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin (IL)-10) in large-vessel endothelial cells (ECs) at levels as high as obtained with the cytomegalovirus (CMV) immediate early promoter, while retaining EC specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely four copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large-vessel EC, while minimizing systemic effects.

Local Vascular Gene Therapy With Apolipoprotein A-I to Promote Regression of Atherosclerosis.

Objective— Gene therapy, delivered directly to the blood vessel wall, could potentially prevent atherosclerotic lesion growth and promote atherosclerosis regression. Previously, we reported that a helper-dependent adenoviral (HDAd) vector expressing apolipoprotein A-I (apoA-I) in carotid endothelium of fat-fed rabbits reduced early (4 weeks) atherosclerotic lesion growth. Here, we tested whether the same HDAd—delivered to the existing carotid atherosclerotic lesions—could promote regression. Approach and Results— Rabbits (n=26) were fed a high-fat diet for 7 months, then treated with bilateral carotid gene transfer. One carotid was infused with an HDAd expressing apoA-I (HDAdApoAI) and the other with a control nonexpressing HDAd (HDAdNull). The side with HDAdApoAI was randomized. Rabbits were then switched to regular chow, lowering their plasma cholesterols by over 70%. ApoA-I mRNA and protein were detected in HDAdApoAI-transduced arteries. After 7 weeks of gene therapy, compared with HDAdNull-treated arteries in the same rabbits, HDAdApoAI-treated arteries had significantly less vascular cell adhesion molecule-1 expression (28%; P=0.04) along with modest but statistically insignificant trends toward decreased intimal lesion volume, lipid and macrophage content, and intercellular adhesion molecule-1 expression (9%–21%; P=0.1–0.4). Post hoc subgroup analysis of rabbits with small-to-moderate–sized lesions (n=20) showed that HDAdApoAI caused large reductions in lesion volume, lipid content, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression (30%–50%; P⩽0.04 for all). Macrophage content was reduced by 30% (P=0.06). There was a significant interaction (P=0.02) between lesion size and treatment efficacy. Conclusions— Even when administered on a background of aggressive lowering of plasma cholesterol, local HDAdApoAI vascular gene therapy may promote rapid regression of small-to-moderate–sized atherosclerotic lesions.

Apo A-I (Apolipoprotein A-I) Vascular Gene Therapy Provides Durable Protection Against Atherosclerosis in Hyperlipidemic Rabbits

Objective— Gene therapy that expresses apo A-I (apolipoprotein A-I) from vascular wall cells has promise for preventing and reversing atherosclerosis. Previously, we reported that transduction of carotid artery endothelial cells with a helper-dependent adenoviral (HDAd) vector expressing apo A-I reduced early (4 weeks) fatty streak development in fat-fed rabbits. Here, we tested whether the same HDAd could provide long-term protection against development of more complex lesions. Approach and Results— Fat-fed rabbits (n=25) underwent bilateral carotid artery gene transfer, with their left and right common carotids randomized to receive either a control vector (HDAdNull) or an apo A-I–expressing vector (HDAdApoAI). Twenty-four additional weeks of high-fat diet yielded complex intimal lesions containing lipid-rich macrophages as well as smooth muscle cells, often in a lesion cap. Twenty-four weeks after gene transfer, high levels of apo A-I mRNA (median ≥250-fold above background) were present in all HDAdApoAI-treated arteries. Compared with paired control HDAdNull-treated arteries in the same rabbit, HDAdApoAI-treated arteries had 30% less median intimal lesion volume (P=0.03), with concomitant reductions (23%–32%) in intimal lipid, macrophage, and smooth muscle cell content (P⩽0.05 for all). HDAdApoAI-treated arteries also had decreased intimal inflammatory markers. VCAM-1 (vascular cell adhesion molecule-1)–stained area was reduced by 36% (P=0.03), with trends toward lower expression of ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein 1), and TNF-&agr; (tumor necrosis factor-&agr;; 13%–39% less; P=0.06–0.1). Conclusions— In rabbits with severe hyperlipidemia, transduction of vascular endothelial cells with an apo A-I–expressing HDAd yields at least 24 weeks of local apo A-I expression that durably reduces atherosclerotic lesion growth and intimal inflammation.

A Rabbit Model for Testing Helper-Dependent Adenovirus-Mediated Gene Therapy for Vein Graft Atherosclerosis

Coronary artery bypass vein grafts are a mainstay of therapy for human atherosclerosis. Unfortunately, the long-term patency of vein grafts is limited by accelerated atherosclerosis. Gene therapy, directed at the vein graft wall, is a promising approach for preventing vein graft atherosclerosis. Because helper-dependent adenovirus (HDAd) efficiently transduces grafted veins and confers long-term transgene expression, HDAd is an excellent candidate for delivery of vein graft-targeted gene therapy. We developed a model of vein graft atherosclerosis in fat-fed rabbits and demonstrated long-term (≥20 weeks) persistence of HDAd genomes after graft transduction. This model enables quantitation of vein graft hemodynamics, wall structure, lipid accumulation, cellularity, vector persistence, and inflammatory markers on a single graft. Time-course experiments identified 12 weeks after transduction as an optimal time to measure efficacy of gene therapy on the critical variables of lipid and macrophage accumulation. We also used chow-fed rabbits to test whether HDAd infusion in vein grafts promotes intimal growth and inflammation. HDAd did not increase intimal growth, but had moderate—yet significant—pro-inflammatory effects. The vein graft atherosclerosis model will be useful for testing HDAd-mediated gene therapy; however, pro-inflammatory effects of HdAd remain a concern in developing HDAd as a therapy for vein graft disease.

190. Strategies for Achieving High-Level, Stable, Endothelium-Specific Transgene Expression Using Helper-Dependent Adenovirus

Endothelial cells (EC) are attractive targets for gene therapy because they can deliver therapeutic proteins both locally and systemically. Helper-dependent adenoviral (HDAd) vectors are useful for EC-targeted gene therapy because they efficiently transduce EC and express transgenes persistently in vivo. However, obtaining high-level and stable in vivo expression from HDAd is a challenge. Our current goal of transducing EC with HDAd to treat atherosclerosis will likely require high-level, stable expression of atheroprotective genes such as apolipoprotein A-I (apoA-I) and interleukin-10 (IL-10).