Rapid and Accurate Diagnosis of Dermatophyte Infections Using the DendrisCHIP® Technology.

Dermatophytosis is a superficial fungal infection with an ever-increasing number of patients. Culture-based mycology remains the most commonly used diagnosis, but it takes around four weeks to identify the causative agent. Therefore, routine clinical laboratories need rapid, high throughput, and accurate species-specific analytical methods for diagnosis and therapeutic management. Based on these requirements, we investigated the feasibility of DendrisCHIP® technology as an innovative molecular diagnostic method for the identification of a subset of 13 pathogens potentially responsible for dermatophytosis infections in clinical samples. This technology is based on DNA microarray, which potentially enables the detection and discrimination of several germs in a single sample. A major originality of DendrisCHIP® technology is the use of a decision algorithm for probability presence or absence of pathogens based on machine learning methods. In this study, the diagnosis of dermatophyte infection was carried out on more than 284 isolates by conventional microbial culture and DendrisCHIP®DP, which correspond to the DendrisCHIP® carrying oligoprobes of the targeted pathogens implicated in dermatophytosis. While convergence ranging from 75 to 86% depending on the sampling procedure was obtained with both methods, the DendrisCHIP®DP proved to identify more isolates with pathogens that escaped the culture method. These results were confirmed at 86% by a third method, which was either a specific RT-PCR or genome sequencing. In addition, diagnostic results with DendrisCHIP®DP can be obtained within a day. This faster and more accurate identification of fungal pathogens with DendrisCHIP®DP enables the clinician to quickly and successfully implement appropriate antifungal treatment to prevent the spread and elimination of dermatophyte infection. Taken together, these results demonstrate that this technology is a very promising method for routine diagnosis of dermatophytosis.

Pathological effects of lung radiofrequency ablation that contribute to pneumothorax, using a porcine model.

OBJECTIVES: The incidence of pneumothorax is 7 times higher after lung radiofrequency ablation (RFA) than after lung biopsy. The reasons for such a difference have never been objectified. The histopathologic changes in lung tissue are well-studied and established for RF in the ablation zone. However, it has not been previously described what the nature of thermal injury might be along the shaft of the RF electrode as it traverses through normal lung tissue to reach the ablation zone. The purpose of this study was to determine the changes occurring around the RF needle along the pathway between the ablated zone and the pleura. MATERIAL AND METHODS: In 3 anaesthetised and ventilated swine, 6 RFA procedures (right and left lungs) were performed using a 14-gauge unipolar multi-tined retractable 3 cm radiofrequency LeVeen probe with a coaxial introducer positioned under CT fluoroscopic guidance. In compliance with literature guidelines, we implemented a gradually increasing thermo-ablation protocol using a RF generator. Helical CT images were acquired pre- and post-RFA procedure to detect and evaluate pneumothorax. Four percutaneous 19-gauge lung biopsies were also performed on the fourth swine under CT guidance. Swine were sacrificed for lung ex vivo examinations, scanning electron microscopy (SEM) and pathological analysis. RESULTS: Three severe (over 50 ml) pneumothorax were detected after RFA. In each one of them, pathological examination revealed a fistulous tract between ablation zone and pleura. No fistulous tract was observed after biopsies. In the 3 cases of severe pneumothorax, the tract was wide open and clearly visible on post procedure CT images and SEM examinations. The RFA tract differed from the needle biopsy tract. The histological changes that are usually found in the ablated zone were observed in the RFA tract's wall and were related to thermal lesions. These modifications caused the creation of a coagulated pulmonary parenchyma rim between the thermo-ablation zone and the pleural space. The structural properties of the damage can explain why the RFA tract is remains patent after needle withdrawal. CONCLUSION: Our study demonstrates for the first time that the changes around the RF needle are the same as in the ablated zone. The damage could create fistulous tracts along the needle path between thermo-ablation zone and pleural space. These fistulas could certainly be responsible for severe pneumothorax that occurs in many patients treated with lung RFA.

A nanoengineered embolic agent for precise radiofrequency ablation.

The purpose of the work is to investigate whether the electromagnetic properties of multi-walled carbon nanotubes (MWCNT) in the presence of radiofrequency (RF) energy is (1) safe, and (2) improves the precision of the therapeutic efficiency of the RF-ablation (RFA) procedure. An in vitro phantom was created for evaluating temperature near RF treated nanotubes. For the in vivo study, three baboons and six pigs were submitted for RFA procedure in superior/inferior kidney poles embolized with a non-adherent, lipophilic embolic agent (marsembol) with or without MWCNT. Tissue damage in the surrounding kill zone was assayed through caspase-3 activation. The in vitro results showed marked heat increase only in the region of the nanotubes. In vivo, necrosis/ischemic damage resulted from RFA therapy alone, RFA plus marsembol only. In marsembol + MWCNT condition, dramatic disruption of cell membranes and sub-cellular organelles was found whereas the nuclear membranes and basal cell membranes remained largely intact. The marsembol vaporized under RFA and tissue fluid filled the space. This caused the MWCNT to cluster within the new aqueous environment. RFA plus marsembol + MWCNT created a well-defined demarcation between healthy and apoptotic cells as evidenced by a marked reduction of caspase-3 expression. By contrast, there was a much less defined ablation zone in the absence of MWCNT. In conclusion, the combination of RFA plus marsembol + MWCNT embolization delineated the kill zone in vitro and in vivo. We demonstrate that MWCNTs remain in the ablation region thus minimizing their migration to the systemic circulation.

Appearance of gas collections after scuba diving death: a computed tomography study in a porcine model.

INTRODUCTION: Postmortem computed tomography can easily demonstrate gas collections after diving accidents. Thus, it is often used to support the diagnosis of air embolism secondary to barotrauma. However, many other phenomenons (putrefaction, resuscitation maneuvers, and postmortem tissue offgassing) can also cause postmortem gas effusions and lead to a wrong diagnosis of barotrauma. OBJECTIVES: The aim of this study is to determine topography and time of onset of postmortem gas collections respectively due to putrefaction, resuscitation maneuvers, and tissue offgassing. MATERIALS AND METHODS: A controlled experimental study was conducted on nine pigs. Three groups of three pigs were studied postmortem by CT from H0 to H24: one control group of nonresuscitated nondivers, one group of divers exposed premortem to an absolute maximal pressure of 5 b for 16 min followed by decompression procedures, and one group of nondivers resuscitated by manual ventilation and thoracic compression for 20 min. The study of intravascular gas was conducted using CT scan and correlated with the results of the autopsy. RESULTS: The CT scan reveals that, starting 3 h after death, a substantial amount of gas is observed in the venous and arterial systems in the group of divers. Arterial gas appears 24 h after death for the resuscitated group and is absent for the first 24 h for the control group. Concerning the putrefaction gas, this provokes intravenous and portal gas collections starting 6 h after death. Subcutaneous emphysema was observed in two of the three animals from the resuscitated group, corresponding to the thoracic compression areas. CONCLUSION: In fatal scuba diving accidents, offgassing appears early (starting from the first hour after death) in the venous system then spreads to the arterial system after about 3 h. The presence of intra-arterial gas is therefore not specific to barotrauma. To affirm a death by barotrauma followed by a gas embolism, a postmortem scanner should be conducted very early. Subcutaneous emphysema should not be mistaken as diagnostic criteria of barotrauma because it can be caused by the resuscitation maneuvers.

Acute effect of a dual ETA-ETB receptor antagonist on pulmonary arterial vasculature in preterm lamb fetuses with surgically induced diaphragmatic hernia.

PURPOSE: To study the effects of tezosentan, a dual ETA and ETB receptor antagonist on the cardiopulmonary profile in a fetal lamb model of CDH in utero. METHODS: A diaphragmatic hernia was surgically created at day 75 of gestation. During 45 min of tezosentan perfusion (1 mg/kg), hemodynamic parameters (pulmonary and aortic pressures, left pulmonary and aortic flows, left auricle pressure, heart rate) were measured at day 135 of gestation. Age-matched fetal lambs served as control animals. Secondarily, parietal tension of vessels rings of pulmonary arteries was assessed in organ baths under increasing concentration of tezosentan. RESULTS: In CDH group, under perfusion of tezosentan, pulmonary artery pressure decreased from 45.8 ± 4.1 to 37.6 ± 5.9 mmHg (P < 0.05). Pulmonary artery flow and pulmonary vascular resistance remained constant. In control group, pulmonary artery flow increased from 153.9 ± 15.8 to 233.4 ± 26 ml/min (P < 0.05). Pulmonary artery pressure did not vary. Subsequently calculated pulmonary vascular resistance decreased. In organ bath, no significant relaxation was observed. CONCLUSION: In this fetal lamb model of CDH, tezosentan decreased pulmonary artery pressure but did not modify pulmonary blood flow. Endothelin may play a role in the regulation of pulmonary vascular tone in utero.

Effectiveness of endovascular embolization with a collagen-based embolic agent (Marsembol) in an animal model.

PURPOSE: To investigate in a porcine experimental model the effectiveness, tissue penetration, and histologic impact of renal artery embolization with a collagen-based nonadhesive embolic agent, marsembol. MATERIALS AND METHODS: Fifteen pigs underwent embolization of one interlobular artery of the renal artery with collagen-resorcinol gel emulsified with Lipiodol and further polymerized with glutaraldehyde-formaldehyde mixture. Angiograms were obtained before, during, and after the procedure. Animals were euthanized at day 0 (n = 3), 1 week (n = 3), or 3 months (n = 7), and flat-panel three-dimensional rotational radiologic images of the kidneys were obtained. Arterial, medullary, and cortical samples were taken for histologic and scanning electron microscopic investigations. RESULTS: Fifteen interlobular renal arteries were successfully embolized by delivering 1.7 mL + or - 0.2 of the embolic agent. All the embolized arteries remained occluded at 3 months, leading to a major atrophy of the embolized portions of the kidneys. Imaging and histologic findings show that the embolic agent provided a distal vessel occlusion and entirely filled the lumen of the arteries up to the glomerular tufts. The homogeneous plug formed by the embolic agent induces very few inflammatory responses. The regenerative tubular processes were arrested at 3 months. CONCLUSIONS: The collagen-based embolic agent described here has the properties required to perform embolization. These specific properties lead to very distal vessel embolization. The embolic agent is effective at 3 months in renal embolization.

Phasic hemodynamics and reverse blood flows in the aortic isthmus and pulmonary arteries of preterm lambs with pulmonary vascular dysfunction.

Time-domain representations of the fetal aortopulmonary circulation were carried out in lamb fetuses to study hemodynamic consequences of congenital diaphragmatic hernia (CDH) and the effects of endothelin-receptor antagonist tezosentan (3 mg/45 min). From the isthmic aortic and left pulmonary artery (PA) flows (Q) and isthmic aortic, PA, and left auricle pressures (P) on day 135 in 10 controls and 7 CDH fetuses (28 ewes), discrete-triggered P and Q waveforms were modelized as Pt and Qt functions to obtain basic hemodynamic profiles, pulsatile waves [P, Q, and entry impedance (Ze)], and P and Q hysteresis loops. In the controls, blood propelling energy was accounted for by biventricular ejection flow waves (kinetic energy) with low Ze and by flow-driven pressure waves (potential energy) with low Ze. Weak fetal pulmonary perfusion was ensured by reflux (reverse flows) from PA branches to the ductus anteriosus and aortic isthmus as reverse flows. Endothelin-receptor antagonist blockade using tezosentan slightly increased the forward flow but largely increased diastolic backward flow with a diminished left auricle pre- and postloading. In CHD fetuses, the static component overrode phasic flows that were detrimental to reverse flows and the direction of the diastolic isthmic flow changed to forward during the diastole period. Decreased cardiac output, flattened pressure waves, and increased forward Ze promoted backward flow to the detriment of forward flow (especially during diastole). Additionally, the intrapulmonary arteriovenous shunting was ineffective. The slowing of cardiac output, the dampening of energetic pressure waves and pulsatility, and the heightening of phasic impedances contributed to the lowering of aortopulmonary blood flows. We speculate that reverse pulmonary flow is a physiological requirement to protect the fetal pulmonary circulation from the prominent right ventricular stream and to enhance blood flow to the fetal heart and brain.

Inhibition of restenosis formation without compromising reendothelialization as a potential solution to thrombosis following angioplasty?

Stent thrombosis remains an important problem after the implantation of different stent types. A potential solution to this problem may be vasoactive agents with dual effects on different cell types like C-type natriuretic peptide (CNP). Therefore, in vitro and in vivo effects of CNP were investigated in a porcine restenotic model. Gene transfer of CNP in cultures of porcine vascular cells revealed up to 30% reduction of growth of smooth muscle cells (p<.05), but no suppression of endothelial growth using CNP. Applied in vivo, angiography revealed a trend of reduced restenosis formation in balloon-injured porcine arteries treated with CNP gene or beta-galactosidase (beta-Gal) control gene after three months (2.59 +/- 2.04-fold reduction, p = n.s.). Histologically, morphometry revealed significantly reduced neointima formation after treatment with CNP plasmid (7.26 +/- 1.44-fold reduction, p < .05). Evans blue staining demonstrated complete endothelial repair already 3 weeks after intervention using CNP. Transfer of CNP gene resulted in a significant inhibition of neointima formation without compromising endothelial repair. Therefore, use of the CNP gene may offer a solution to suppress restenosis formation while preventing subacute or late thrombosis.

Meta-analysis shows similar risk of thrombosis after drug-eluting stent, bare-metal stent, or angioplasty.

Coronary stent thrombosis remains an important problem after the implantation of different stent types. This study investigates the risk of stent thrombosis associated with the use of drug-eluting stents (DESs), bare-metal stents (BMSs) compared to balloon angioplasty. A meta-analysis of 28 randomized trials involving 5612 versus 7639 versus 2994 patients with coronary heart disease treated with DES, BMS, or balloon angioplasty was therefore performed. Comparing the implantation of DES versus BMS, DES was not found to increase the hazard for thrombosis up to 15 months (odds ratio [OR] = 0.86, 95% confidence interval [CI] 0.58 to 1.3, p < .48). There was also no significant difference in the hazard for subacute thrombosis (SAT) or late stent thrombosis (LST) in the DES versus BMSs group (OR = 0.86, 95% CI 0.50 to 1.5, p < .6 and OR = 0.92, 95% CI 0.50 to 1.68, p < .78, respectively). Comparing incidences of stent thromboses in patients receiving balloon angioplasty or implantation of BMS, the rate of SAT in the balloon angioplasty group (1.7% SAT) versus BMS group (1.8% SAT) was also similar (OR = 0.93, 95% CI 0.61 to 1.4, p < .71). Finally, there was no significant difference in the occurrence of stent thrombosis for the different coatings of DESs. In conclusion, the use of DES was not observed to have a significant effect on stent thrombosis events, compared with the implantation of BMS or balloon angioplasty.

Functional cerebral venous outflow in swine and baboon: feasibility of an intracranial venous hypertension model.

INTRODUCTION: To evaluate the feasibility of performing a functional cerebral venous outflow blockage in two large animals species, the swine and the baboon, for elaboration of venous hypertension models. METHOD: Cerebral venous outflow pathways were identified on angiogram and venography of three swine and two baboons, and potential approaches to access these structures were assessed. Practicability of performing functional intracranial dural outflow blockage was tested. RESULTS: The main cerebral venous outflow route was the internal jugular vein in baboons and the paraspinal venous network in swine. Both animals had an additional venous outflow structure, the petrosquamous sinus. Access to intracranial venous sinuses was achieved through a percutaneous retrograde approach in baboon but not in swine, due to the absence of a direct connection between the dural structures and the internal jugular vein. A transcranial approach allowed to access dural venous structures in swine. In both models, partial and progressive venous sinus occlusion increased intracranial pressure, while preserving the animal's vital status. At 6 months, all animals are alive with no neurological deficits. CONCLUSION: Functional venous dural outflow blockage for elaboration of intracranial venous hypertension is feasible in both models. To be effective, the sinus blockage must be performed before the origin of the petrosquamous, an additional venous sinus seen in swine and baboon. The baboon has the greatest advantage of resembling human cerebral venous drainage, which enables an intracranial venous retrograde access. However, the transcranial approach remains a valuable option to access intracranial venous sinuses in swine.

Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo.

The potential of two small poly-L-lysines (sPLLs), low molecular weight sPLL (LMW-L) containing 7-30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer (GT) with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N(5)-carbamoyl-spermine)-propane] in vascular smooth muscle cells (SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA (50-100 nm). At high ionic strength, large complexes (>1 microm) were observed without any significant differences in particle size between lipoplexes (DOCSPER/DNA) and lipopolyplexes (DOCSPER/sPLL/DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/sPLL/DNA formulations enhanced GT in vitro up to 5-fold, in a porcine model using local periadventitial application up to 1.5-fold. Both sPLLs significantly increased liposome-mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery.

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.

Sevoflurane alters right ventricular performance but not pulmonary vascular resistance in acutely instrumented anesthetized pigs.

OBJECTIVE: Although the effects of halogenated agents on both normal and diseased left ventricles have been widely studied, the influence of these anesthetic agents on right ventricular (RV) performance remains less well characterized. This study was undertaken to examine the effects of 2 different concentrations of sevoflurane on RV function, and coronary and pulmonary hemodynamics in acutely instrumented anesthetized pigs. DESIGN: Prospective experimental study. SETTING: Laboratory of experimental research in a university teaching hospital. SUBJECTS: Anesthetized pigs. INTERVENTIONS: Regional RV function in 10 pigs was determined from pressure segment length loop analysis, global RV function from stroke work versus end-diastolic pressure relation, right coronary blood flow, and pulmonary vascular resistance (PVR), without and then with 2.6% (minimum alveolar concentration [MAC]) and 3.9 % (1.5 MAC) end-tidal sevoflurane concentrations. MAIN RESULTS: Sevoflurane preserved inflow systolic shortening and RV regional external work, but significantly depressed outflow systolic shortening (p < 0.05). Global RV stroke work was depressed to 72% +/- 12% and 61% +/- 10% of baseline value, respectively, with 1 and 1.5 MAC of sevoflurane (p < 0.05), but without alteration of PVR. Right coronary blood flow decreased dose dependently. CONCLUSIONS: Sevoflurane causes significant depression of global RV function associated with a qualitatively different effect on inflow and outflow tracts, without any modification of PVR.

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.

Balloon-pump-induced pulsatility improves coronary and carotid flows in an experimental model of BioMedicus left ventricular assistance.

The aim of this study is to evaluate the benefit of the simultaneous use of a BioMedicus left ventricular assistance device (Medtronic, Minneapolis, MN, U.S.A.) and an intra-aortic balloon pump on regional blood flows, pressure, and pulsatility. Twelve pigs are studied. A BioMedicus pump was placed between the left atrium and the ascending aorta and an intra-aortic balloon pump was inserted through the left femoral artery. Blood flow and pressure were measured in the carotid, femoral, and coronary arteries and in the thoracic aorta below the intra-aortic balloon in the basal experimental condition with a full-flow BioMedicus pump and with a full-flow BioMedicus pump + intra-aortic balloon. The BioMedicus pump eliminates pulsatility in all sites and significantly decreases coronary and carotid blood flow. The adjunction of an intra-aortic balloon restores pulsatility to values comparable to those recorded in basal conditions. Coronary and carotid flows even increase to values higher than in the basal conditions. The simultaneous use of an intra-aortic balloon combined with the BioMedicus pump provides a pulsatile flow and increases coronary and carotid blood flows in pigs. An intra-aortic balloon can easily be combined with a BioMedicus pump whenever possible and may improve myocardial recovery in patients with postcardiotomy ventricular failure.

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.