Thrombomodulin overexpression to limit neointima formation.

BACKGROUND-These studies were initiated to confirm that high-level thrombomodulin overexpression is sufficient to limit neointima formation after mechanical overdilation injury. METHODS AND RESULTS-An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on neointima formation 28 days after mechanical overdilation injury was evaluated. New Zealand White rabbit common femoral arteries were treated with buffer, viral control, or Adv/RSV-THM and subjected to mechanical overdilation injury. The treated vessels (n=4 per treatment) were harvested after 28 days and evaluated to determine intima-to-media (I/M) ratios. Additional experiments were performed to determine early (7-day) changes in extracellular elastin and collagen content; local macrophage, T-cell, and neutrophil infiltration; and local thrombus formation as potential contributors to the observed impact on 28-day neointima formation. The construct significantly decreased neointima formation after mechanical dilation injury in this model. By histological analysis, buffer controls exhibited mean I/M ratios of 0.76+/-0.06%, whereas viral controls reached 0.77+/-0.08%; in contrast, Adv/RSV-THM reduced I/M ratios to 0.47+/-0.06%. Local inflammatory infiltrate decreased in the Adv/RSV-THM group relative to controls, whereas matrix remained relatively preserved. Rates of early thrombus formation also decreased in Adv/RSV-THM animals. CONCLUSIONS-This construct thus offers a viable technique for promoting a locally neointima-resistant small-caliber artery via decreased thrombus bulk, normal matrix preservation, and decreased local inflammation without the inflammatory damage that has limited many other adenoviral applications.

Therapeutic elastase inhibition by alpha-1-antitrypsin gene transfer limits neointima formation in normal rabbits.

PURPOSE: Alpha-1-antitrypsin (AAT) is the major circulating elastase inhibitor. Deficiency of elastase inhibition leads to emphysema and vascular abnormalities including accelerated neointima. Because recent evidence suggests that tissue AAT levels determine inhibitory function, the authors hypothesize that local tissue-based expression of AAT limits elastase activity sufficiently to guide arterial response to injury. MATERIALS AND METHODS: Rabbit common femoral arteries were injured by mechanical overdilation and treated with buffer, viral control, or an adenovirus expressing AAT (Ad/AAT). After 3 and 28 days, intima-to-media (I/M) ratios were evaluated. Additionally, early changes in elastase inhibition potential (3 d), extracellular elastin and collagen content (3 d), and local macrophage and neutrophil infiltration (7 d) were determined. RESULTS: Ad/AAT significantly decreased neointima formation after mechanical dilation injury after 28 days: buffer controls exhibited mean I/M ratios of 0.76 +/- 0.06, whereas viral controls reached 0.77 +/- 0.09; in contrast, Ad/AAT reduced I/M ratios to 0.44 +/- 0.06. Both early elastin and collagen content were preserved in the Ad/AAT group relative to controls. The Ad/AAT group also reversed the local inflammation that characterized viral controls. CONCLUSIONS: This strategy demonstrates that local increases in elastase inhibition potential promote a neointima-resistant small-caliber artery, which may offer new promise in management of patients undergoing angioplasty.

Enhancement of neointima formation with tissue-type plasminogen activator.

PURPOSE: Indirect evidence suggests that tissue plasminogen activator (tPA) either limits or does not alter restenosis. However, tPA enhances tumor invasiveness through matrix remodeling, and several elements of degraded matrix enhance smooth muscle cell mitogenesis. We use either local adenoviral-mediated overexpression of tPA or systemic infusion of recombinant tPA combined with mechanical overdilation of rabbit common femoral arteries to evaluate the impact of tPA on neointima formation. METHODS: Left common femoral arteries of New Zealand white rabbits were transfected in situ either with an adenoviral-construct-expressing tPA or a viral control (adenoviral-construct-expressing beta-galactosidase) or nonviral (buffer) control after balloon angioplasty injury. At 7 and 28 days, left common femoral artery segments were harvested (n = 4 for each group and time point). Vessel segments were examined for intimato-media ratio, smooth muscle cell proliferation, extracellular matrix, and inflammatory response. Thrombus formation was evaluated after 3 days (n = 3 for each group). In a second experiment, New Zealand white rabbits (n = 3 per group, per time point) underwent mechanical dilation followed by buffer treatment or systemic tPA infusion according to a widely clinically used accelerated infusion protocol. Treated artery segments were harvested after 7 or 28 days and processed for intima-to-media ratio determination and class-wide histochemical determination of collagenous extracellular matrix and collagen content. RESULTS: Both rate and degree of neointima formation increase dramatically with overexpression (250%-461% relative to controls at 7 and 28 days). Substantial early matrix degradation is observed in vessels treated with local overexpression of tPA, although no increases in local inflammation or in smooth muscle proliferation occur. Late enhancement of smooth muscle proliferation emerges, consistent with secondary impact of perturbed matrix components. Systemic infusion of tPA according to clinical protocols also results in early and late enhancement of neointima formation in this model (34%-52% relative to controls at at 7 and 28 days), with significant early collagenous matrix degradation. Systemic infusion, although significant, did not attain the degree of neointima formation present with overexpression. CONCLUSION: With some evidence of dose-dependence, tissue plasminogen activator enhances neointima formation after angioplasty in a rabbit model. Early matrix degradation precedes change in rates of proliferation and underlies this effect in spite of several antirestenotic actions including decreased thrombus and decreased macrophage recruitment in this model.