Evidence of hypoxic areas within the arterial wall in vivo

The anoxemia theory of atherosclerosis states that an imbalance between the demand and supply of oxygen in the arterial wall is a key factor for the development of atherosclerotic lesions. Direct in vitro and in situ measurements have shown that PO2 is decreased in the more deeply situated parts of the media, but the degree of hypoxia in vivo or the distribution of hypoxia along the arterial tree is not known. For this reason, we have developed a method for the detection of hypoxia in the arterial wall in vivo by using a hypoxia marker, 7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline, that may be visualized by immunofluorescence. In the present study, we have used this method in rabbits with experimentally induced atherosclerosis. Our results indicate that zones of hypoxia occur at depth in the atherosclerotic plaque. The mechanism was probably an impaired oxygen diffusion capacity due to the thickness of the lesion, together with high oxygen consumption by the foam cells. Thus, we have for the first time demonstrated that hypoxia actually does exist in the arterial wall in vivo, lending support to the anoxemia theory of atherosclerosis.     

Adaptation to arterial wall hypoxia demonstrated in vivo with oxygen microcathodes

Iliofemoral arteries of 9 rabbits were balloon de-endothelialized resulting in subintimal thickening. Contrary to expectation, enzyme and lactate determinations did not indicate arterial wall hypoxia when compared with arteries of 10 control rabbits. The explanation came from in vivo measurement of oxygen tension profiles across the de-endothelialized and control femoral arteries and from the subsequent histological findings. They showed that the impaired oxygen supply of the de-endothelialized arteries with subintimal thickening was counteracted by a centripetal oxygenation of the arterial wall obviously induced by proliferation of newly formed nutrient vessels in the adventitia. Such adaptation is an important mechanism against hypoxia induced by arterial injury and may be an essential protective factor in atherogenesis.     

Sustained local delivery of heparin to the rabbit arterial wall with an electroporation catheter

Current methods of local drug delivery frequently fail to achieve a prolonged therapeutically effective tissue drug level without producing vascular trauma. A novel double-balloon catheter system, incorporating electroporation technology, has been designed and tested to deliver heparin into rabbit carotid arteries in an overstretch balloon injury model in vivo. Following arterial injury, fluoresceinated heparin was delivered into the volume between the two inflated balloons, and the artery was subjected to an electrical pulse. Catheter deployment and endovascular electrical pulsing were well-tolerated in all animals (N = 21) without adverse hemodynamic and histological changes. Periodic arterial blood samples revealed no abnormalities in the clotting profile or any gross morphological changes in the blood cells up to 8 hr after treatment. Much stronger heparin fluorescence was detected throughout the vessel layers for at least 12 hr in the pulsed samples compared to the control. Histochemical staining of the tissue showed intracellular localization of heparin. Endovascular electroporation may provide better retention and higher therapeutic efficacy than can be achieved by conventional systemic delivery of heparin at clinically safe concentrations.Cathet. Cardiovasc. Diagn. 45:337–345, 1998. © 1998 Wiley-Liss, Inc.     

Ankle-Brachial index by oscillometry: A very useful method to assess peripheral arterial disease in diabetes

Background:

Peripheral Arterial Disease (PAD) remains the least recognized form of atherosclerosis. The Ankle-Brachial Index (ABI) has emerged as one of the potent markers of diffuse atherosclerosis, cardiovascular (CV) risk, and overall survival in general public, especially in diabetics. The important reason for the lack of early diagnosis is the non-availability of a test that is easy to perform and less expensive, with no training required.

Objectives:

To evaluate the osillometric method of performing ABI with regard to its usefulness in detecting PAD cases and to correlate the signs and symptoms with ABI.

Materials and Methods:

Two hundred diabetics of varying duration attending the clinic for a period of eight months, from August 2006 to April 2007, were evaluated for signs, symptoms, and risk factors. ABI was performed using the oscillometric method. The positives were confirmed by Doppler evaluation. An equal number of age- and sex-matched controls, which were ABI negative, were also assessed by Doppler. Sensitivity and Specificity were determined.

Results:

There were 120 males and 80 females. Twelve males (10%) and six females (7.5%) were ABI positive. On Doppler, eleven males (91.5%) and three females (50%) were true positives. There were six false negatives from the controls (three each). The Sensitivity was 70% and Specificity was 75%. Symptoms and signs correlated well with ABI positives. Hypertension was the most important risk factor.

Conclusions:

In spite of the limitations, the oscillometric method of performing ABI is a simple procedure, easy to perform, does not require training and can be performed as an outpatient procedure not only by doctors, but also by the paramedical staff to detect more PAD cases.     

A method for in-line measurement of lumen and wall of microscopic vessels in vivo

A system incorporating an image-splitter eyepiece to the television microscope and monitoring units, allows accurate measurements and rapid analogue recording of microvessel geometry in vivo. The measurement of dynamic changes of lumen size and wall thickness is made by shearing the projected split vessel image in the T.V. monitor. The output of a sensitive potentiometer incorporated in the micrometer head of the splitter registers the amount of image-shearing into an absolute measurement, reproducible to 0.1% on the polygraph. Simultaneous measurements of inner and outer radii of target microvessels can be made at any azimuth in the microscopic field, up to ×3000 magnification of optico-electrical image. Higher magnifications, up to ×6500, and maximum resolution, permitted shearing of selected images of structures anatomically and functionally corresponding to smooth muscle cells in the wall of microvessels. The numerical information obtained permitted estimation of dynamic changes of lumen and wall cross-sectional areas and the relation of lumen to wall from moment to moment with great accuracy in vivo.     

A fluorescent in vivo microscopic method to assess surface mucosal integrity in the rat stomach. Effects of ethanol and prostaglandin

A new sensitive in vivo fluorescent method to assess gastric mucosal integrity in the anesthetized rat is described. Topically applied fluorescein diacetate enters gastric mucosal cells. The diacetate is cleaved by intracellular esterases leaving fluorescein trapped within the cells. The pattern of fluorescence can be visualized, and the intensity of fluorescence measured, using a fluorescent in vivo microscopy system. Frozen section studies revealed that fluorescein was located in the surface mucous cells and the mucous neck cells. Topically applied ethanol caused a dose-dependent decline in intensity of fluorescence. Measurement of fluorescence in the supernatant bathing the mucosa revealed that leak of fluorescein out of cells or shedding of cells was, at least in part, responsible for the decline in fluorescence intensity. Pretreatment with a "cytoprotective" dose of 16,16-dimethyl prostaglandin E2 did not protect against the decline in fluorescence seen after 12.5% and 25% ethanol. This confirms findings of previous histologic studies that prostaglandin "cytoprotection" does not include surface cell protection. We conclude that this technique provides a sensitive, quantitative in vivo method to study gastric surface cell injury.     

A general method to assess similarity of protein structures, with applications to T4 bacteriophage lysozyme.

A method is proposed that permits the structural similarity between any pair of proteins to be analyzed in a completely general manner. In the proposed procedure, all possible structural segments of a given length from one protein are compared with all possible segments from the other protein. This set of comparisons reveals any structural similarities between the two proteins being compared, and also provides a basis for estimating the probability that a particular degree of structural homology could have occurred by chance. Application of the method to the comparison of T4 bacteriophage lysozyme and carp calcium-binding protein suggests that the previously reported structural similarity between parts of these two proteins [Tufty, R. M.& Kretsinger, R. H. (1975) Science 187, 167-169] is no better than would be expected by chance. On the other hand, the structural correspondence between phage lysozyme and hen egg-white lysozyme [Rossman, M.G. & Argos, P. (1976) J. Mol. Biol. 105, 75-96] does appear to be significant.     

Feasibility of very-high resolution ultrasound to assess elastic and muscular arterial wall morphology in adolescents attending an outpatient clinic for obesity and lipid abnormalities

Objective

Atherosclerosis begins during early life and is accelerated in individuals with cardiovascular risk factors. We hypothesized that very-high resolution ultrasound (VHRU, 25–55 MHz) could feasibly detect early arterial changes in adolescents with risk factors.

Methods

We prospectively imaged the carotid, brachial and radial arterial morphology (far wall intima–media thickness, IMT; adventitia thickness, AT) by VHRU in 58 youths (age 14 ± 2 years) attending a Pediatric Preventive Cardiology Clinic for assessment and management of cardiovascular risk factors and compared the findings to those from an age-matched group of 67 controls.

Results

Brachial and radial imaging was successful for all subjects. The carotid far wall could not be imaged in 7% of the patients due to limitations in penetration. VHRU image quality was related to body size and imaging depth. Imaging and analysis time were 12 ± 3 and 18 ± 3 min, respectively. Carotid IMT was increased in patients (0.42 ± 0.05 vs. 0.40 ± 0.06 mm, p = 0.05). No differences were found in brachial or radial IMT or AT vs. controls. Age, male gender, body mass index, systolic blood pressure (BP), but not lipid levels, were associated with arterial IMT in regression analyses.

Conclusion

VHRU is feasible in imaging carotid and peripheral muscular artery IMT in adolescents. The arterial IMT is associated with age, gender, adiposity and systolic BP, but not lipid levels, in this adolescent population. Further studies including patients with manifest clinical atherosclerosis are needed to assess if VHRU has applications in atherosclerosis research.     

Polylysine as a vehicle for extracellular matrix-targeted local drug delivery, providing high accumulation and long-term retention within the vascular wall

We present the first steps in the elaboration of an approach of extracellular matrix-targeted local drug delivery (ECM-LDD), designed to provide a high concentration, ubiquitous distribution, and long-term retention of a drug within the vessel wall after local intravascular delivery. The approach is based on the concept of a bifunctional drug comprising a "therapeutic effector" and an "affinity vehicle," which should bind to an abundant component of the vessel wall. The aim of the present study was to select molecules suitable for the role of affinity vehicles for ECM-LDD and to study their intravascular delivery and retention ex vivo and in an animal model. By use of fluorescence microscopy, the following molecules were selected on the basis of strong binding to cross sections of human vessels: protamine, polylysine, polyarginine, a glycosaminoglycan-binding peptide from vitronectin, and a synthetic dendrimer. With polylysine as a prototypic affinity vehicle, we showed that after intravascular delivery, polylysine was concentrated throughout a luminal layer of the vascular wall to an extremely high concentration of 20 g/L and was retained therein for at least 72 hours of perfusion without noticeable losses. Low molecular weight (fluorescein) and high molecular weight (hirudin) compounds could be chemically conjugated to polylysine and were retained in the vessel wall after intravascular delivery of the conjugates. In conclusion, by use of the ECM-LDD method, an extremely high concentration and long-term retention of locally delivered drug can be reached. Polycationic molecules can be considered as potential affinity vehicles for ECM-LDD.     

猪整体和局部心室复极离散度的单相动作电位标测比较

目的评价整体心室复极离散度（d ispersion of ventricu lar repolarization,DVR）能否从心内膜几个邻近点或几个远距离点的标测来估测。方法应用CARTO标测系统,在10头猪左心室心内膜的（75±12）个位点记录单相动作电位。计算每一点复极结束时间（end-of-repolarization tim e,EORT）和动作电位时程（monophasic action potentialduration,MAPD）。关于EORT和MAPD的整体DVR与相应的局部DVR进行比较。局部DVR包括面积在2 cm2内的邻近DVR;还包括左室最早和最晚激动点间的远距离DVR1以及左室心尖部和外基底部间的远距离DVR2。结果关于EORT和MAPD的邻近DVR[（15±4）m s和（12±4）m s]显著小于相应的整体DVR[（84±31和77±26）m s,P<0.01]。远距离DVR1[（42±19和23±14）m s]和远距离DVR2[（25±16和18±11）m s]显著大于邻近DVR（P<0.01）,但仍显著小于整体DVR（P<0.01）。结论从心内膜几个邻近点或几个远距离点的标测不能良好地估测整体DVR。在估测整体DVR中获取整体信息很重要。     

Rupture of the arterial wall causes deflection in pressure time course during ex vivo balloon angioplasty

A relation between restenosis and arterial lesions resulting from balloon angioplasty has been suggested in literature. Nevertheless, it is unclear to what extent angioplasty-induced arterial wall lesions contribute to the occurrence of restenosis. One problem is that arterial ruptures cannot be detected during balloon inflation. This study describes a method to detect ruptures in the arterial wall, based on deflections observable in the development of the balloonpressure. We performed ex vivo angioplasty with constant strain rate on 28 human femoral artery segments, showing deflections in 21 cases. In 20 cases wall rupture was confirmed histologically. From seven cases not showing deflections, four showed intact wall at microscopy. These figures result in a selectivity of the proposed method of 87 ± 7% and a predictive value of the positive test of 95 ± 5%. We conclude that this method can enhance detection of arterial rupture during ex vivo angioplasty and may become important clinically. Cathet. Cardiovasc. Diagn. 42:92–101, 1997. © 1997 Wiley-Liss, Inc.     

Validation of methods to assess potential biomarkers in pediatric patients with esophageal eosinophilia

AIM: To validate methods for determining mast cell density, extracellular major basic protein content, and presence of fibrosis in esophageal eosinophilia.METHODS: Twenty specimens with > 20 eosinophils/high-power field (hpf) classified as high eosinophil density (HE) and 20 specimens with < 5 eosinophils/hpf classified as low esophageal density (LE) were identified. All 40 specimens underwent immunohistochemical staining and trichrome staining. Mast cell density, extracellular major basic protein (MBP) density, and presence of subepithelial fibrosis were assessed in a standardized manner. All specimens were evaluated by two separate observers and by a single observer on two separate occasions to evaluate reproducibility of the methods.RESULTS: A strong inter-observer correlation was noted for both peak and mean mast cell counts (r = 0.725, P < 0.0001 and r = 0.823, P < 0.0001). A strong intra-observer correlation also was noted for both peak and mean mast cell counts (r = 0.752, P < 0.0001 and r = 0.878, P < 0.0001). A very strong inter-observer correlation was noted for both peak (τ = 0.867, P < 0.0001) and mean extracellular MBP densities (r = 0.925, P < 0.0001). A very strong intra-observer correlation was noted for both peak (τ = 0.875; P < 0.0001) and mean extracellular MBP densities (r = 0.956, P < 0.0001). Excellent inter-rater reliability was found for fibrosis (κ = 0.887). Mast cell and MBP densities, as well as presence of fibrosis, were significantly increased in HE vs LE. The HE group had significantly higher intraepithelial mast cell peak (29.35 ± 21.61 vs 12.45 ± 8.26, P = 0.002) and mean (19.84 ± 15.81 vs 6.35 ± 4.5, P = 0.001) densities than the LE group. The HE group had significantly higher peak extracellular MBP (2.35 ± 0.67 vs 0.45 ± 0.61, P < 0.001) and mean extracellular MBP (1.95 ± 0.76 vs 0.20 ± 0.29, P < 0.0001) densities than the LE group. Seventy-three percent of patients with HE (11/15) had fibrosis, whereas only 10% of patients with LE (1/10) had fibrosis (P < 0.01). MBP performed the best in predicting classification of HE vs LE, with mean MBP demonstrating 100% sensitivity and 95% specificity at the optimal cut point.CONCLUSION: This study provides methodology and proof-of-concept for future evaluation of these biomarkers for differentiating esophageal eosinophilic diseases such as reflux esophagitis and eosinophilic esophagitis.