Targeted gene transfer of hepatocyte growth factor to alveolar type II epithelial cells reduces lung fibrosis in rats

Inefficient alveolar wound repair contributes to the development of pulmonary fibrosis. Hepatocyte growth factor (HGF) is a potent growth factor for alveolar type II epithelial cells (AECII) and may improve repair and reduce fibrosis. We studied whether targeted gene transfer of HGF specifically to AECII improves lung fibrosis in bleomycin-induced lung fibrosis. A plasmid encoding human HGF expressed from the human surfactant protein C promoter (pSpC-hHGF) was designed, and extracorporeal electroporation-mediated gene transfer of HGF specifically to AECII was performed 7 days after bleomycin-induced lung injury in the rat. Animals were killed 7 days after hHGF gene transfer. Electroporation-mediated HGF gene transfer resulted in HGF expression specifically in AECII at biologically relevant levels. HGF gene transfer reduced pulmonary fibrosis as assessed by histology, hydroxyproline determination, and design-based stereology compared with controls. Our results indicate that the antifibrotic effect of HGF is due in part to a reduction of transforming growth factor-β(1), modulation of the epithelial-mesenchymal transition, and reduction of extravascular fibrin deposition. We conclude that targeted HGF gene transfer specifically to AECII decreases bleomycin-induced lung fibrosis and may therefore represent a novel cell-specific gene transfer technology to treat pulmonary fibrosis.

Maternal cholestasis during pregnancy programs metabolic disease in offspring

The intrauterine environment is a major contributor to increased rates of metabolic disease in adults. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that affects 0.5%-2% of pregnant women and is characterized by increased bile acid levels in the maternal serum. The influence of ICP on the metabolic health of offspring is unknown. We analyzed the Northern Finland birth cohort 1985-1986 database and found that 16-year-old children of mothers with ICP had altered lipid profiles. Males had increased BMI, and females exhibited increased waist and hip girth compared with the offspring of uncomplicated pregnancies. We further investigated the effect of maternal cholestasis on the metabolism of adult offspring in the mouse. Females from cholestatic mothers developed a severe obese, diabetic phenotype with hepatosteatosis following a Western diet, whereas matched mice not exposed to cholestasis in utero did not. Female littermates were susceptible to metabolic disease before dietary challenge. Human and mouse studies showed an accumulation of lipids in the fetoplacental unit and increased transplacental cholesterol transport in cholestatic pregnancy. We believe this is the first report showing that cholestatic pregnancy in the absence of altered maternal BMI or diabetes can program metabolic disease in the offspring.

Quantification of Popliteal Artery Deformation During Leg Flexion in Subjects With Peripheral Artery Disease: A Pilot Study

Purpose: To quantify the in vivo deformations of the popliteal artery during leg flexion in subjects with clinically relevant peripheral artery disease (PAD). Methods: Five patients (4 men; mean age 69 years, range 56–79) with varying calcification levels of the popliteal artery undergoing endovascular revascularization underwent 3-dimensional (3D) rotational angiography. Image acquisition was performed with the leg straight and with a flexion of 70°/20° in the knee/hip joints. The arterial centerline and the corresponding branches in both positions were segmented to create 3D reconstructions of the arterial trees. Axial deformation, twisting, and curvatures were quantified. Furthermore, the relationships between the calcification levels and the deformations were investigated. Results: An average shortening of 5.9%±2.5% and twist rate of 3.8±2.2°/cm in the popliteal artery were observed. Maximal curvatures in the straight and flexed positions were 0.12±0.04 cm−1 and 0.24±0.09 cm−1, respectively. As the severity of calcification increased, the maximal curvature in the straight position increased from 0.08 to 0.17 cm−1, while an increase from 0.17 to 0.39 cm−1 was observed for the flexed position. Axial elongations and arterial twisting were not affected by the calcification levels. Conclusion: The popliteal artery of patients with symptomatic PAD is exposed to significant deformations during flexion of the knee joint. The severity of calcification directly affects curvature, but not arterial length or twisting angles. This pilot study also showed the ability of rotational angiography to quantify the 3D deformations of the popliteal artery in patients with various levels of calcification.

Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.

Prognostic relevance of coronary collateral function: confounded or causal relationship?

OBJECTIVE To expand the limited information on the prognostic impact of quantitatively obtained collateral function in patients with coronary artery disease (CAD) and to estimate causality of such a relation. DESIGN Prospective cohort study with long-term observation of clinical outcome. SETTING University Hospital. PATIENTS One thousand one hundred and eighty-one patients with chronic stable CAD undergoing 1771 quantitative, coronary pressure-derived collateral flow index measurements, as obtained during a 1-min coronary balloon occlusion (CFI is the ratio between mean distal coronary occlusive pressure and mean aortic pressure both subtracted by central venous pressure). Subgroup of 152 patients included in randomised trials on the longitudinal effect of different arteriogenic protocols on CFI. INTERVENTIONS Collection of long-term follow-up information on clinical outcome. MAIN OUTCOME MEASURES All-cause mortality and major adverse cardiac events. RESULTS Cumulative 15-year survival rate was 48% in patients with CFI<0.25 and 65% in the group with CFI≥0.25 (p=0.0057). Cumulative 10-year survival rate was 75% in patients without arteriogenic therapy and 88% (p=0.0482) in the group with arteriogenic therapy and showing a significant increase in CFI at follow-up. By proportional hazard analysis, the following variables predicted increased all-cause mortality: age, low CFI, left ventricular end-diastolic pressure and number of vessels with CAD. CONCLUSIONS A well-functioning coronary collateral circulation independently predicts lowered mortality in patients with chronic CAD. This relation appears to be causal, because augmented collateral function by arteriogenic therapy is associated with prolonged survival.

G-CSF induced arteriogenesis in humans: molecular insights into a randomized controlled trial

AIMS Recent data have demonstrated the feasibility of therapeutic induction of coronary collateral growth (arteriogenesis); however, mechanisms of action of such therapeutic collateral stimulation in humans are unknown. The aim of this study was to evaluate potential mechanisms, especially the involvement of arteriogenesis-relevant genes. METHODS AND RESULTS A total of 52 patients were randomized into two groups: subcutaneous G-CSF (10 μg/kg; n=26) or placebo (n=26). Before and after this 2-week treatment, collateral-flow index (CFI) was determined by simultaneous measurement of mean aortic, distal coronary occlusive and central venous pressure. CD34+ endothelial progenitor cells (EPC) and monocytes were quantified before, during and after treatment; gene-expression analysis of monocytes was performed with real-time polymerase chain reaction (RT-PCR). G-CSF lead to a significant increase of EPC and monocytes (4.8 and 2.6 fold, p < 0.05); for both cell types, the extent of increase correlated with CFI increase (r=0.23 and 0.14, p < 0.05). G-CSF also induced a change in gene expression of pro-and anti-arteriogenic genes in monocytes. Among nine assessed genes, three were found to be differentially regulated (IL8, JAK2, and PNPLa4; p < 0.05). CONCLUSIONS The mechanism of induction of collateral growth by G-CSF is related to an increase of EPC and of peripheral monocytes. It also leads to a change toward a pro-arteriogenic gene expression in peripheral monocytes.

Distinct roles of estrogen receptors alpha and beta mediating acute vasodilation of epicardial coronary arteries.

This study investigated the contribution of estrogen receptors (ERs) alpha and beta for epicardial coronary artery function, vascular NO bioactivity, and superoxide (O(2)(-)) formation. Porcine coronary rings were suspended in organ chambers and precontracted with prostaglandin F(2alpha) to determine direct effects of the selective ER agonists 4,4',4''-(4-propyl-[(1)H]pyrazole-1,3,5-triyl)tris-phenol (PPT) or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or the nonselective ER agonist 17beta-estradiol. Indirect effects on contractility to U46619 and relaxation to bradykinin were assessed and effects on NO, nitrite, and O(2)(-) formation were measured in cultured cells. Within 5 minutes, selective ERalpha activation by PPT, but not 17beta-estradiol or the ERbeta agonist DPN, caused rapid, NO-dependent, and endothelium-dependent relaxation (49+/-5%; P<0.001 versus ethanol). PPT also caused sustained endothelium- and NO-independent vasodilation similar to 17beta-estradiol after 60 minutes (72+/-3%; P<0.001 versus ethanol). DPN induced endothelium-dependent NO-independent relaxation via endothelium-dependent hyperpolarization (40+/-4%; P<0.01 versus ethanol). 17beta-Estradiol and PPT, but not DPN, attenuated the responses to U46619 and bradykinin. All of the ER agonists increased NO and nitrite formation in vascular endothelial but not smooth muscle cells and attenuated vascular smooth muscle cell O(2)(-) formation (P<0.001). ERalpha activation had the most potent effects on both nitrite formation and inhibiting O(2)(-) (P<0.05). These data demonstrate novel and differential mechanisms by which ERalpha and ERbeta activation control coronary artery vasoreactivity in males and females and regulate vascular NO and O(2)(-) formation. The findings indicate that coronary vascular effects of sex hormones differ with regard to affinity to ERalpha and ERbeta, which will contribute to beneficial and adverse effects of hormone replacement therapy.

Effects of obesity on endothelium-dependent reactivity during acute nitric oxide synthase inhibition: modulatory role of endothelin.

This study investigated vascular reactivity in response to acetylcholine, in the presence of acute inhibition of nitric oxide synthase, in the carotid artery and aorta of obese C57Bl6/J mice fed on a high-fat diet for 30 weeks, and of control mice. A subgroup of obese animals was also treated with the ET(A) receptor antagonist darusentan (50 mg x kg(-1) x day(-1)). In vascular rings from control animals, acetylcholine caused endothelium-dependent contractions in the carotid artery, but not in the aorta. In vascular rings from obese mice, contractility to acetylcholine was also evident in the aorta, and that in the carotid artery was increased compared with control mice. ET(A) receptor blockade by darusentan treatment of the obese mice prevented enhanced vasoconstriction to acetylcholine, resulting in mild vasodilatation. Thus obesity increases endothelium-dependent vasoconstriction in the absence of endothelial nitric oxide. This effect can be completely prevented by chronic ET(A) receptor blockade, suggesting that endothelin modulates increased endothelium-dependent vasoconstriction in obesity.

Mechano-Biologie von bovinen Bandscheiben belastet unter Kompression und Torsion

Einleitung: Bandscheiben wirken als Schockabsorbierer in der Wirbelsäule und auf diese wirken meistens komplexe Kräfte, zusammengesetzt aus Kompression, Torsion und Flexion. Die biomechanishe Umgebung einer Bandscheibe ist denn auch geprägt von komplexen Belastungen. Die Forschung über die in vitro Bandscheibenbiologie hat sich bisher um die axiale Kompression konzentriert, wobei die Bedeutung von Torsion und insbesondere dem Zusammenspiel von Kompression und Torsion (="Twisting") praktisch noch nie untersucht wurde an lebenden Organkultur-Explantaten. Wir präsentieren neue mechanobiologische Daten über die Überlebenswahrscheinlichkeit von Bandscheibenzellen kultiviert in einem neuartigen, kompakten Design eines bi-axialen Bioreaktors, um die Bedeutung von Kompression und Torsion zu verstehen. Material/Methode: Bovine Schwanzbandscheiben mit den Endplatten wurden isoliert wie bereits beschrieben [2] und mechanische Belastung wurde angewendet mit einem 2 DoF Bioreaktor für 14 Tage [3]. Die Bandscheiben wurden in verschiedene Belastungsgruppen eingeteilt: 1) Keine Belastung (NL), 2) zyklische Kompression (CC) [8h: axiale Kompression mit 0.6 ± 0.2 MPa, 0.2 Hz], 3) zyklische Torsion (CT) [8h: ± 2° torsion, 0.2 Hz, 0.2 MPa compression], 4) zyklische Kompression und Torsion (CCT) [8h: 0.6 ± 0.2 MPa, 0.2 Hz & ± 2° torsion, 0.2 Hz]. Das Bandscheibengewebe wurde mit LIVE/DEAD gefärbt und miteinem konfokalen Mikroskop visualisiert um die Überlebensrate zu bestimmen. Zell Apoptosis wurde quantifiziert mit einem Caspase 3/7 Kit normalisiert zum totalen Proteingehalt (Bradford). Relative Gen-Expression von wichtigen Genen für die Bandscheibe wurde bestimmt von anabolischen, katabolischen und inflammatorischen Genen mittels real-time RT-PCR. Die Morphologie der Bandscheibenzellen wurde mittels Histologie bestimmt. Ergebnisse: Die Resultate zeigten einen starken Abfall der Zellüberlebenswahrscheinlichkeit im Zentrum der Bandscheiben, dem Nulceus Pulposus (NP), i.e. 10%, in der Gruppe mit CCT. Hingegen die Überlebenswahrscheinlichkeit im Annulus fibrosus (AF) war stabilisiert bei über 60% im NP und im AF in allen anderen Gruppen (Fig 1). Apoptotische Aktivität war statistisch signifikant erhöht in der CC-Gruppe, aber nicht in der CCT-Gruppe, was die Vermutung nahe legt, dass der erhöhte Zellverlust im NP nicht mit Apoptose sondern mit Nekrose erklärt werden kann. Die Gen Expression der anabolischen Gene COL1, COL2 und Biglycan war signifikant erhöht im AF in der CCT Gruppe, ebenfalls waren Remodeling-Gene angeschaltet wie ADAMTS4 und MMP-13 in der CCT Gruppe (Fig. 2). Der Glykosaminoglykan (GAG) Gehalt war generell im AF erhöht in den Gruppen unter mechanischer Belastung, jedoch nicht statistisch signifikant. Schlussfolgerung: Zyklische Torsion kombiniert mit zyklischer Kompression waren in dieser Studie erfolgreich und nach unserem besten Wissen zum ersten Mal an Bandscheibenexplantaten in einer 14- tägigen Organkultur angewendet worden in einem dafür speziell konzipierten Bioreaktor. Die Resultezeigten überraschend einen negativen Effekt bei physiologischen Parametern, was die Belastung (0.6MPa ± 0.2MPa) und die Torsion (± 2°) angeht. Dieser negative Effekt des "Twistings" auf die Überlebenswahrscheinlichkeit der Zellen war jedoch nur regional im NP von Bedeutung, wohingegen im AF keine Effekte zu detektieren waren.

Intracoronary Optical Coherence Tomography and Histology of Overlapping Everolimus-Eluting Bioresorbable Vascular Scaffolds in a Porcine Coronary Artery Model

OBJECTIVES: This study sought to assess the vascular response of overlapping Absorb stents compared with overlapping newer-generation everolimus-eluting metallic platform stents (Xience V [XV]) in a porcine coronary artery model. BACKGROUND: The everolimus-eluting bioresorbable vascular scaffold (Absorb) is a novel approach to treating coronary lesions. A persistent inflammatory response, fibrin deposition, and delayed endothelialization have been reported with overlapping first-generation drug-eluting stents. METHODS: Forty-one overlapping Absorb and overlapping Xience V (XV) devices (3.0 × 12 mm) were implanted in the main coronary arteries of 17 nonatherosclerotic pigs with 10% overstretch. Implanted coronary arteries were evaluated by optical coherence tomography (OCT) at 28 days (Absorb n = 11, XV n = 7) and 90 days (Absorb n = 11, XV n = 8), with immediate histological evaluation following euthanasia at the same time points. One animal from each time point was evaluated with scanning electron microscopy alone. A total of 1,407 cross sections were analyzed by OCT and 148 cross sections analyzed histologically. RESULTS: At 28 days in the overlap, OCT analyses indicated 80.1% of Absorb struts and 99.4% of XV struts to be covered (p < 0.0001), corresponding to histological observations of struts with cellular coverage of 75.4% and 99.6%, respectively (p < 0.001). Uncovered struts were almost exclusively related to the presence of "stacked" Absorb struts, that is, with a direct overlay configuration. At 90 days, overlapping Absorb and overlapping XV struts demonstrated >99% strut coverage by OCT and histology, with no evidence of a significant inflammatory process, and comparable % volume obstructions. CONCLUSIONS: In porcine coronary arteries implanted with overlapping Absorb or overlapping XV struts, strut coverage is delayed at 28 days in overlapping Absorb, dependent on the overlay configuration of the thicker Absorb struts. At 90 days, both overlapping Absorb and overlapping XV have comparable strut coverage. The implications of increased strut thickness may have important clinical and design considerations for bioresorbable platforms.

Estimation of tool pose based on force-density correlation during robotic drilling

The application of image-guided systems with or without support by surgical robots relies on the accuracy of the navigation process, including patient-to-image registration. The surgeon must carry out the procedure based on the information provided by the navigation system, usually without being able to verify its correctness beyond visual inspection. Misleading surrogate parameters such as the fiducial registration error are often used to describe the success of the registration process, while a lack of methods describing the effects of navigation errors, such as those caused by tracking or calibration, may prevent the application of image guidance in certain accuracy-critical interventions. During minimally invasive mastoidectomy for cochlear implantation, a direct tunnel is drilled from the outside of the mastoid to a target on the cochlea based on registration using landmarks solely on the surface of the skull. Using this methodology, it is impossible to detect if the drill is advancing in the correct direction and that injury of the facial nerve will be avoided. To overcome this problem, a tool localization method based on drilling process information is proposed. The algorithm estimates the pose of a robot-guided surgical tool during a drilling task based on the correlation of the observed axial drilling force and the heterogeneous bone density in the mastoid extracted from 3-D image data. We present here one possible implementation of this method tested on ten tunnels drilled into three human cadaver specimens where an average tool localization accuracy of 0.29 mm was observed.