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

BACKGROUND: 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. METHODS: For in vitro applications, primary cultures of porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were used. Gene transfer was performed with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N5-carbamoylspermine)propane]. In vivo treatment of pig femoral arteries was adventitial using a needle injection catheter following balloon angioplasty. Arteries were investigated by angiography, Evan's blue staining, histomorphometry, immunohistochemistry, PCR and RT-PCR. RESULTS: Using CNP gene transfer in vitro, 29.4+/-7.2% reduction of cell proliferation in VSMCs was observed. In ECs, the CNP gene did not compromise cellular growth. For the CNP peptide the optimal concentration was 1 mM with 50.7+/-11.3% reduction of VSMC proliferation and 12.1+/-5.3% enhancement of growth of ECs. Three weeks following application in vivo complete re-endothelialization was observed in all treated groups. At 3 months significant reduction of neointima formation was observed using CNP gene vs. CNP peptide (85.9+/-7.8% vs. 63.3+/-27.6% reduction, P<0.05) compared to control treatment. CONCLUSION: Periadventitial liposome-mediated CNP gene transfer in vivo resulted in a significant long-term reduction of neointima formation without compromising endothelial repair and was superior over single CNP peptide administration. Advantages of CNP are its physiological origin and simultaneous inhibition of VSMC proliferation and promotion of EC growth.

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 beta-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 beta-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 beta-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 beta-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.

Functional analysis of genomic DNA, cDNA, and nucleotide sequence of the mature C-type natriuretic peptide gene in vascular cells.

OBJECTIVE: The aim of this study was to investigate the effect of various C-type natriuretic peptide (CNP) sequences (genomic DNA [CNPDNA], cDNA derived from mRNA [CNPcDNA], and sequence coding for 22 amino acids of the mature CNP [CNP22aa]) on the growth of primary porcine vascular cells. METHODS AND RESULTS: Gene transfer was performed with cationic lipid DOCSPER or linear polyethylenimine. All 3 CNP sequences led to significant inhibition of smooth muscle cell (SMC) proliferation. In contrast, significant stimulation of cell growth was observed in endothelial cells (ECs) using CNPDNA or CNPcDNA but not CNP22aa. In a porcine restenosis model, a significant reduction in neointima hyperplasia was found 3 months after application of the CNPcDNA vector compared with the control transfection. CONCLUSIONS: The results demonstrate that the first intron in the CNP sequence does not contain any additional enhancer-binding sites. However, the signal sequence is indispensable for secretion of CNP and its appropriate physiological function. Furthermore, the results show for the first time the therapeutic effect of CNP using liposome-mediated gene transfer and local adventitial delivery. Advantages of the CNP gene are its dual effects with inhibition of SMC proliferation and simultaneous promotion of EC growth. Functional analysis of various C-type natriuretic peptide (CNP) sequences on growth of vascular cells. For the first time, dual therapeutic effects of CNP with inhibition of smooth muscle cell proliferation and stimulation of re-endothelialization were demonstrated in a pig restenosis model using liposome-mediated gene transfer and local adventitial delivery.

Comparison of surgical versus endovascular occlusion models in pig femoral arteries.

PURPOSE: To compare an endovascular technique with a well established surgical approach to achieve long-term occlusions of large porcine arteries while preserving the integrity of periarterial tissue. METHODS: The femoral arteries in 11 pigs were occluded using surgical techniques on one side and blinded stent-grafts in the contralateral vessel. Feasibility, safety, primary and long-term success, and the extent of vascularization were determined over a 3-month period by conventional angiography and histological analysis. A subgroup of animals (n=5) was treated with a locally administered plasmid coding for vascular endothelial growth factor (pVEGF165) to compare both occlusion techniques under conditions of collateral growth induction. RESULTS: The primary and long-term success rates for both occlusion models were 100%. Surgical occlusion of arteries resulted in a significant amount of scar dehiscence and local groin infection compared to the endograft-occluded side. There was no significant difference in capillary densities and collateralization of periarterial areas in a comparison of the occlusion technique: the cross-sectional area of the superficial femoral artery (SFA) was 300 +/- 24 mm2 for endovascular occlusion versus 320 +/- 23 mm2 for surgical occlusion (p=0.559). In the profunda femoris artery, respective values were 418 +/- 35 and 448 +/- 18 mm2 (p=0.474). The local delivery of pVEGF165 resulted in a significant increase in collateral growth in both occlusion models with comparable neovascularization: cross-sectional SFA area increased from 310 +/- 16 to 428 +/- 13 mm2 (p<0.0001); in the PFA, the area increased from 422 +/- 19 to 658 +/- 49 mm2 (p<0.0001). CONCLUSIONS: Endovascular arterial occlusions using blinded stent-grafts allow easy and safe creation of long-term occlusions. Previously described collateralization following surgical occlusions was not observed, indicating that those collaterals may be associated with wound healing rather than ischemia. The occlusion of arteries using blinded stent-grafts in pigs may therefore be an appropriate model for assessing the effects of angiogenic factors in vivo.

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.

Pressure-flow loops and instantaneous input impedance in the thoracic aorta: another way to assess the effect of aortic bypass graft implantation on myocardial, brain, and subdiaphragmatic perfusion.

BACKGROUND: The serious disturbances in ventriculoarterial coupling after thoracic aorta bypass grafting are addressed through aortic entry impedance in the frequency domain from flow-pressure waves. We designed a method for synthesizing pressure and flow waves to evaluate opposal to aortic flow along the cardiac cycle, addressing myocardial, brain, and visceral tissue perfusions from pressure-flow hysteresis loops and forward-backward aortic entry impedance in the ascending aorta, transverse aortic arch, and distal descending aorta, respectively, before and after extra-anatomic grafting of the descending aorta in the swine. METHODS: Twelve pigs underwent extra-anatomic grafting (woven double-velour prosthesis, 18-mm diameter), bypassing the descending aorta. Periarterial flow and endovascular pressure signals were mathematically synthesized (error minimization) to yield continuous functions of flow, pressure along the cardiac cycle before treatment for mean hemodynamics, pressure-flow hysteresis loops, and aortic entry impedance. RESULTS: Grafting of the descending aorta overshadowed pressure-flow hysteresis loops in the ascending aorta by shortening maximum pressure delay on maximum flow and diastolic flow reversal. Clamping of the descending aorta substantially restored hemodynamics in the ascending aorta, although the diastolic flow decrease was accelerated. Identical processes developed in the transverse aorta. Subdiaphragmatic descending aortic flow was flattened after grafting and restored, although thickened, after clamping of the descending aorta. Flow wave peak was framed by a diastolic aortic entry impedance peak, which was damped along the transverse aortic arch (aortic entry impedance peak in the ascending aorta, 1700 +/- 102 kN x s x m(-5); aortic entry impedance peak in the descending aorta, 292 +/- 45 kN x s x m(-5); P <.05). After grafting, the aortic entry impedance peak was transferred to early systole (aortic entry impedance peak in the transverse aortic arch, 2104 +/- 94 kN x s x m(-5); aortic entry impedance peak in the descending aorta, 450 +/- 75 kN x s x m(-5); P <.05). Clamping of the descending aorta attenuated the early systolic aortic entry impedance peak (aortic entry impedance peak in the transverse aortic arch, 1269 +/- 104 kN x s x m(-5); aortic entry impedance peak in the descending aorta, 491 +/- 75 kN x s x m(-5); P <.05), although aortic entry impedance in the descending aorta remained higher than before grafting (P <.05). Specifically, the backward flow ascending aorta to coronary trunks generated a backward aortic entry impedance peak (2234 +/- 350 kN x s x m(-5)) superimposed onto the forward aortic entry impedance peak with asymptotic boundaries that diminished after grafting and further enlarged after clamping of the descending aorta. CONCLUSIONS: Hemodynamic opposition of grafting of the descending aorta are specific to the aortic site and cardiac cycle and are dependent on clamping of the descending aorta. Our approach to thoracic aorta hemodynamics could enable optimization of bypass grafting.

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.

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.

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.

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.

Vascular endothelial growth factor (VEGF165) and its influence on angiogenesis versus arteriogenesis in different vascular beds.

PURPOSE: To use local gene delivery to determine any district-specific influence of vascular endothelial growth factor (VEGF(165)) on angiogenesis and arteriogenesis in arteries of distinct developmental origin. METHODS: Coronary and peripheral arteries were chronically occluded in 30 Pietrain pigs using a percutaneous approach and blinded stent-graft. DNA was delivered to the adventitia in dosages corresponding to 10% of the body weight-adapted amount used in clinical trials. The coronary arteries in 12 animals and the peripheral arteries in 12 animals were treated or used as controls (no occlusion or occlusion with transfection of the beta-galactosidase gene). Six additional animals were sacrificed at 1 or 3 weeks for expression analyses, while the other 24 animals were sacrificed at 5 months for expression analysis and histology. Angiography, polymerase chain reaction analyses, and immunohistochemistry were performed. RESULTS: Expression of the VEGF gene was observed at 1 and 3 weeks following application, while transfected DNA was detected up to 5 months. New collaterals formed around occluded coronary arteries (2.63 +/- 0.69 fold, p<0.05 versus 1.24 +/- 0.40 fold for peripheral arteries), and angiographic arterial area increase was more pronounced in coronary (2.49 +/- 0.59 fold, p<0.05) than peripheral arteries (1.49 +/- 0.05 fold). There was no collateralization surrounding occluded peripheral arteries, but new arterial branches were seen (2.0 +/- 0.28, p<0.05 versus 1.07 +/- 0.31 for coronary). CONCLUSIONS: The response to VEGF, whether it is predominantly angiogenesis or arteriogenesis, is dependent on the target vessel. These observed differences in the behavior of arteries may be related to their differing developmental origins, which may have important implications for future therapeutic strategies using VEGF in different vessels.