Pierre H. Rolland

Hemodynamics and Wall Mechanics of a Compliance Matching Stent: In Vitro and In Vivo Analysis

PURPOSE Evidence is emerging that the abrupt compliance mismatch that exists at the junction between the stent ends and the host arterial wall disturbs both the vascular hemodynamics and the natural wall stress distribution. These stent-induced alterations are greatly reduced by smoothing the compliance mismatch between the stent and host vessel. A stent that provides this smooth transition in compliance, the compliance matching stent (CMS), has been developed. This study attempts to evaluate the hemodynamics and wall mechanical consequences of the CMS both in vitro and in vivo. MATERIALS AND METHODS Finite element analysis was used to assess the solid mechanical behavior (compliance and stress) of the CMS in a stent/artery hybrid structure. A similar analysis was performed with a Palmaz stent. In vivo hemodynamics and wall mechanical changes induced by the CMS were investigated in a swine model from direct measurements of flow, pressure, diameter, and histology in the stented segment of superficial femoral arteries after 7 days. RESULTS Finite element analysis showed that the abrupt compliance mismatch was substantially smoothed between the vessel portions with and without the stent with CMS segments. Circumferential stress was also markedly reduced with the CMS compared to other stent. The in vivo results showed that the CMS was efficient in compliance matching and did not dampen flow or pressure waves downstream the stent. Concurrent histology showed limited thrombus and inflammatory cell accumulation around the stent struts. CONCLUSION These results indicate that the stent/artery hybrid structure can be compliance matched with proper stent design and that this structure limits solid mechanical stress and hemodynamic disturbances. It remains to be seen whether compliance-matched vascular stents reduce in-stent restenosis.

Alteration in prostacyclin and prostaglandin E2 production. Correlation with changes in human aortic atherosclerotic disease.

Prostacyclin (PGI2) and prostaglandin E2 (PGE2) production was Investigated in human aortas (five controls and 27 with atherosclerotic lesions). The specific activities of PGI2 and PGE2 synthetase were studied using radioimmunoassays of PGE2 and 6-keto-PGE1α, of aortic microsomes incubated in the presence of additional substrate and cofactors. The atherosclerotic lesions were examined under the light microscope and were classified as Stage 1 when the disease was restricted to the intima and as Stages 2 and 3 when there were moderate or advanced lesions. Prostaglandin production for the control group (n = 5), Stage 1 (n = 7), Stage 2 (n = 10), and Stage 3 (n = 10) were as follows: 454 ± 15, 162 ± 81, 92 ± 90, and 65 ± 61 pmol 6-K-PGF1α /50 mg proteln/10 minutes; and 15 ± 12, 399 ± 406, 227 ± 174, and 366 ± 362 pmol PGE2/50 mg protein/10 minutes (mean ± SD) respectively. We conclude that: 1) In normal aortas, PGE2 production was low, while PGI2 synthesis activity was elevated. The reverse situation was observed In aortas with atherosclerosis lesions (p < 0.05). 2) There was an inverse relationship between PGE2 and PGI1 production (p < 0.05). 3) There was a direct hlstologlc relationship between lower PGI2 production and atherosclerosis progression. A decided decline In 6-K-PGF1α production was detected in aortas In the early stages (65% of control values). 4) By contrast, a progressive increase In PGE2 production was found In Stage 2 and Stage 3 groups (p < 0.05). These results demonstrate that there are correlations between the changes In prostaglandin production and the morphological features of atherosclerosis development. Because of the pharmacologlc properties of prostaglandins, these changes in prostaglandin production may promote the development of atherosclerosis.

Homocysteine induces synthesis of a serine elastase in arterial smooth muscle cells from multi-organ donors

OBJECTIVES In heart transplant recipients with diffuse coronary arteriopathy, we have previously demonstrated the prevalence of elevated homocysteinemia, also known as an independent risk factor for myocardial infarction and stroke. In hyperhomocysteinemic mini-pigs we also observed early detectable pathologic changes in the elastic laminae. We hypothesized that homocysteine causes premature breakdown in the arterial elastic fibers by activation of the elastolytic activities. METHODS We examined the effect of homocysteine on elastase-like production by smooth muscle cells from sub-inguinal arteries of multi-organ donors (23.4 +/- 3.4 yr, n = 8). The freshly isolated cells were incubated for 0-72 h with homocysteine (0-250 microM), in the presence or absence of specific protease inhibitors. RESULTS Homocysteine was devoid of a direct effect, but after 18 h incubation the elastase-like activities increased by 5-6-fold in the extracellular medium. The enzymes were characterized as serine proteases. Incubation of cells with a nucleic acid synthesis inhibitor (actinomycin D) or a protein synthesis inhibitor (cycloheximide) suppressed the enzyme induction. CONCLUSIONS This is the first report of serine protease induction by homocysteine in vascular smooth muscle cells. The process may require protein synthesis and account for the early alterations of the arterial elastic structures in heart transplant recipients, and in other hyperhomocysteinemic patients, as well.

Association of mild hyperhomocysteinemia with cardiac graft vascular disease

In non-transplant patients mild hyperhomocysteinemia is an independent risk factor for vascular disease. The aim of this study was to determine whether hyperhomocysteinemia is associated with graft vascular disease. Fasting total plasma homocysteine was assessed in 18 patients with graft vasculopathy and 18 transplanted patients without graft vasculopathy matched for age, sex and the time since transplant. All were on cyclosporin. Graft vasculopathy was defined at coronary angiography as stenoses > or = 25%, or aneurysms. We found that hyperhomocysteinemia ( > or = 15 micromol/l) is common among transplanted heart recipients and significantly more frequent in the patients with graft vasculopathy (17/18 versus 11/18). Accordingly, the mean homocysteinemia was significantly higher in the group with graft vasculopathy (23.6+/-7.8 versus 16.9+/-7.1 micromol/l, P=0.01). The elevation of homocysteine plasma levels in the heart transplant recipients has probably multiple causes. The main cause seems to be renal failure. Additional causes could be azathioprine treatment or genetic polymorphisms. These results suggest that besides the immunological factors, homocysteine can play an additional role in the pathogenesis of graft vascular disease.

Optimization of nonviral transfection: variables influencing liposome-mediated gene transfer in proliferating vs. quiescent cells in culture and in vivo using a porcine restenosis model.

Abstract. Cationic liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays, PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation compared with the transfection using a control gene for ß-galactosidase. Thus, in this vascular model, initial optimization of lipofection in stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling, low toxicity, and the lack of carcinogenicity or immunogenic reactions.

Evaluation of Endovascular Techniques for Creating a Porcine Femoral Artery Occlusion Model

Purpose: To determine the optimal endovascular approach to achieve long-term occlusion of large arteries, while preserving the integrity of periarterial tissue, in an animal model of ischemia. Methods: Femoral artery occlusions were created in 16 pigs using detachable balloons, coils, or blinded stent-grafts. Feasibility, safety, primary and long-term success, and the degree of neovascularization were determined over a 6-month period by serial angiography and histological analyses. Four animals served as untreated controls. Results: Overall primary success for all occlusion devices was 100%. The 6-month occlusion rate using detachable balloons or coils was 33% and 0%, respectively; however, all arteries occluded with blinded stent-grafts remained obstructed to the end of the study. There was no significant difference in capillary densities and collateralization of periarterial areas when occluded arteries were compared with nonoccluded controls in the same animal. No increase in collateralization was observed following endovascular arterial occlusion. Conclusions: Percutaneous insertion of blinded stent-grafts easily, safely, and reliably creates long-term arterial occlusion in pigs, which may make this a more appropriate model for studying the effects of angiogenic factors in vivo.

Efficacy of Local Molsidomine Delivery from a Hydrogel-Coated Angioplasty Balloon Catheter in the Atherosclerotic Porcine Model

Purpose: To evaluate the therapeutic effects of local molsidomine delivery via a hydrogel-coated angioplasty balloon catheter during overstretch angioplasty in atherosclerotic swine iliac vessels. Molsidomine is retained in the arterial wall after local delivery for more than 72 hr and is slowly metabolized into linsidomine, releasing nitric oxide (NO). Methods: A hydrogel-coated angioplasty balloon catheter was used to both deliver drug locally (150 mg molsidomine or placebo in the contralateral vessel) and dilate iliac vessels in nine Pietrin pigs that had been on an atherogenic diet for 5 months. Animals were killed at 3 hr (n = 2), 24 hr (n = 3) and 3 months (n = 3) after treatment. Iliac arteries were examined for wall pulsatility, histomorphometry, cell proliferation and platelet aggregation. Results: No significant therapeutic effects were detected 3 hr after treatment. At 24 hr, wall pulsatility, thromboresistance and vascular cell homeostasis were significantly restored in the molsidomine-treated versus placebo group. At 3 months, molsidomine inhibited restenotic lesion development, except in scarred areas of histologically detectable adventitial/medial dissection. Conclusion: Local delivery of concentrated molsidomine from a hydrogel-coated angioplasty balloon catheter resulted in early NO-dependent vasodilation/stress normalization and antithrombotic and antiproliferative effects. In the medium term, molsidomine inhibited restenosis in the absence of vessel dissection.

C-type natriuretic peptide for reduction of restenosis: gene transfer is superior over single peptide administration.

Restenosis is still a significant clinical problem limiting the long‐term therapeutic success following balloon dilation or stent implantation. New approaches are necessary inhibiting neointima formation and simultaneously promoting re‐endothelialization. Therefore, long‐term therapeutic effects of adventitial liposome‐mediated C‐type natriuretic protein (CNP) gene and CNP peptide applications in a porcine model for restenosis post‐angioplasty were investigated.

Vascular Endothelial Growth Factor Response in Porcine Coronary and Peripheral Arteries Using Nonsurgical Occlusion Model, Local Delivery, and Liposome-Mediated Gene Transfer

Angiogenesis and arteriogenesis play an important role in advanced vascular occlusive diseases. Whether angiogenesis or arteriogenesis predominate depends on the preexisting collateral vessel network, the type and location of occlusion, and different developmental origin of the arteries. Angiogenesis and arteriogenesis were investigated following vascular endothelial growth factor (VEGF) treatment in different arteries important in occlusive arterial diseases using a newly developed porcine arterial occlusion model. Porcine coronary and peripheral arteries were occluded interventionally using blinded stent grafts. Gene transfer was performed using a needle injection catheter and cationic lipid DOCSPER as gene carrier. DNA and gene expression in arterial tissue was examined using polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR. Vessel development was determined by angiography, immunohistochemistry, and measurement of capillary density. The transfected gene and its expression were found 3 months following application. In tissue adjacent to coronary arteries, there was significantly enhanced capillary density but no increase in angiographic score. In contrast, tissue surrounding peripheral arteries demonstrated no enhancement of capillary density but an enhancement in angiographic score. These results demonstrate differential responses to VEGF treatment in coronary and peripheral arteries resulting predominantly in either angiogenesis or arteriogenesis. Further investigation of VEGF signaling pathway is necessary for better understanding of the processes of vascular development, which may have potential impact on the design of cardiovascular therapeutics.

C-Type Natriuretic Peptide Inhibits Constrictive Remodeling without Compromising Re-Endothelialization in Balloon-Dilated Renal Arteries

Purpose: To investigate the long-term effect of local, liposome-mediated gene transfer of C-type natriuretic peptide (CNP) plasmid versus CNP protein on restenosis in porcine renal arteries following balloon angioplasty. Methods: The renal arteries of 15 pigs were dilated and the adventitia at the site of balloon injury injected with CNP protein, pCR3.1 plasmid encoding CNP, or the β-galactosidase gene (control) via a needle injection catheter. Five animals receiving the CNP and control genes in dilated arteries were sacrificed after 3 weeks to analyze re-endothelialization, proliferation, and early CNP expression. Ten animals designated for the long-term experiments (3 months) were treated with the CNP gene versus CNP protein (n=3), the CNP gene versus the control gene (n=3), and the CNP protein versus the control gene (n=3). One animal served as a dilated non-treated control. Transfection and expression of CNP and β-galactosidase were measured by polymerase chain reaction (PCR) and reverse transcription PCR. Renal arterial lumen narrowing was measured with angiography and histology. Endothelialization was assessed using Evans blue stain; vWF, CD31, factor VIII, and Ki67 were markers for immunohistochemical analysis. Results: An intact endothelial layer was seen at 3 weeks following angioplasty in all transfected arteries. Three months following treatment, computer-assisted morphometric analysis revealed significant enlargement of the arterial cross-sectional areas in CNP plasmid-treated vessels compared to dilated but untreated arteries (CNP plasmid +34.8%±13.9% versus CNP protein −1.75%±19.9% versus β-galactosidase −47.0%±13.9%, p < 0.01). Angiographic analysis showed significant enlargement of the arterial diameter compared to dilated, untreated arteries (CNP plasmid +20.8%±6.8% versus CNP protein +5.7%±6.0% versus β-galactosidase −24.5%±10.2%, p < 0.01). Conclusions: Local application of CNP plasmid proved superior to CNP protein in producing rapid re-endothelialization and significantly enlarging the renal arterial lumen following dilation.