Smooth muscle cells of penis in the rat: noninvasive quantification with shear wave elastography.

PURPOSE: Smooth muscle cells (SMCs) of cavernosum play an important role in erection. It is of great significance to quantitatively analyze the level of SMCs in penis. In this study, we investigated the feasibility of shear wave elastography (SWE) on evaluating the level of SMCs in penis quantitatively. MATERIALS AND METHODS: Twenty healthy male rats were selected. The SWE imaging of penis was carried out and then immunohistochemistry analysis of penis was performed to analyze the expression of alpha smooth muscle actin in penis. The measurement index of SWE examination was tissue stiffness (TS). The measurement index of immunohistochemistry analysis was positive area percentage of alpha smooth muscle actin (AP). RESULTS: Sixty sets of data of TS and AP were obtained. The results showed that TS was significantly correlated with AP and the correlation coefficient was -0.618 (p < 0.001). The result of TS had been plotted against the AP measurements. The relation between the two results has been fitted with quadric curve; the goodness-of-fit index was 0.364 (p < 0.001). CONCLUSIONS: The level of SMCs in penis was successfully quantified in vivo with SWE. SWE can be used clinically for evaluating the level of SMCs in penis quantitatively.

Synthesis and characterization of transiently stable albumin-coated microbubbles via a flow-focusing microfluidic device.

We describe a method for synthesizing albumin-shelled, large-diameter (>10 µm), transiently stable microbubbles using a flow-focusing microfluidic device (FFMD). The microfluidic device enables microbubbles to be produced immediately before insonation, thus relaxing the requirements for stability. Both reconstituted fractionated bovine serum albumin (BSA) and fresh bovine blood plasma were investigated as shell stabilizers. Microbubble coalescence was inhibited by the addition of either dextrose or glycerol and propylene glycol. Microbubbles were observed to have an acoustic half-life of approximately 6 s. Microbubbles generated directly within a vessel phantom containing flowing blood produced a 6.5-dB increase in acoustic signal within the lumen. Microbubbles generated in real time upstream of in vitro rat aortic smooth muscle cells under physiologic flow conditions successfully permeabilized 58% of the cells on insonation at a peak negative pressure of 200 kPa. These results indicate that transiently stable microbubbles produced via flow-focusing microfluidic devices are capable of image enhancement and drug delivery. In addition, successful microbubble production with blood plasma suggests the potential to use blood as a stabilizing shell.

Non-invasive assessment of elastic modulus of arterial constructs during cell culture using ultrasound elasticity imaging.

Mechanical strength is a key design factor in tissue engineering of arteries. Most existing techniques assess the mechanical property of arterial constructs destructively, leading to sacrifice of a large number of animals. We propose an ultrasound-based non-invasive technique for the assessment of the mechanical strength of engineered arterial constructs. Tubular scaffolds made from a biodegradable elastomer and seeded with vascular fibroblasts and smooth muscle cells were cultured in a pulsatile-flow bioreactor. Scaffold distension was computed from ultrasound radiofrequency signals of the pulsating scaffold via 2-D phase-sensitive speckle tracking. Young's modulus was then calculated by solving the inverse problem from the distension and the recorded pulse pressure. The stiffness thus computed from ultrasound correlated well with direct mechanical testing results. As the scaffolds matured in culture, ultrasound measurements indicated an increase in Young's modulus, and histology confirmed the growth of cells and collagen fibrils in the constructs. The results indicate that ultrasound elastography can be used to assess and monitor non-invasively the mechanical properties of arterial constructs.

Imaging and analyzing the elasticity of vascular smooth muscle cells by atomic force acoustic microscope.

Vascular smooth muscle cells (VSMCs) play an important role in the good performance of the vasculature. To study the surface, intracellular structure and elasticity of VSMCs, atomic force acoustic microscope (AFAM) was used for imaging VSMCs from A7r5 rat aorta arteries. The topography images of VSMCs were obtained in contact mode and the acoustic images were obtained by AFAM in sample vibration mode. Then, the force curve measurement derived using Young's modulus of the interested areas was used for evaluating elasticity properties. The acoustic images were found in higher resolution with more information than the topography images. The force curves showed the difference in Young's modulus of the different parts of VSMC. These findings demonstrate that AFAM is useful for displaying the surface, structure and elasticity property of VSMCs clearly, with short scanning time, negligible harm or damage to cell and nanometer-level resolution.

Enhanced antiproliferative effects of combination hexokinase II shRNA and NIS gene therapy on vascular smooth muscle cells.

INTRODUCTION: This study was designed to determine the antiproliferative effects of combination gene therapy using sodium iodide symporter (NIS)-based radioiodine and lentivirus-mediated short hairpin RNA (shRNA) against hexokinase II (HKII) on vascular smooth muscle cells (VSMCs). METHODS: A7r5 rat VSMCs were stably transfected with a dual-expression vector of NIS and Fluc (A7r5-NL cells). Functional assessment was performed by radioiodine uptake assay, luciferase assay and confocal microscopy. After exposure to lentivirus-HKII-shRNA, the (18)F-FDG uptake test and HK activity assay were performed. The effects of combination therapy with (131)I and lentivirus-HKII-shRNA on VSMCs were assessed with an in vitro clonogenic assay. In vivo bioluminescence and nuclear imaging were undertaken using a xenografted mouse model. RESULTS: In vitro functional assessment confirmed expression of NIS and Fluc genes in A7r5-NL, but not in parent A7r5 cells. Transfection of lentivirus-HKII-shRNA resulted in a significant decrease in messenger RNA expression of the HKII gene, (18)F-FDG uptake and HK activity. The cell survival rate of A7r5-NL decreased to 61.9% and 90.5% by single therapy with 7.4 MBq of (131)I or lentivirus-HKII-shRNA, respectively, and further decreased to 42.9% by combined therapy (P<.05). In vivo bioluminescent and gamma camera images clearly demonstrated optical signals and (99m)Tc pertechnetate uptake at the site of A7r5-NL cell inoculation in nude mice. CONCLUSION: The enhanced antiproliferative effect on VSMCs was achieved by a combination of NIS-based radioiodine and lentivirus-mediated HKII shRNA gene therapy. Successful demonstration of in vivo dual reporter gene imaging assures the potential for further application in an animal model.

Lymphangioleiomyomatosis: what do we know and what are we looking for?

Lymphangioleiomyomatosis (LAM) is a rare disease characterised by proliferation of abnormal smooth muscle-like cells (LAM cells) leading to progressive cystic destruction of the lung, lymphatic abnormalities and abdominal tumours. It affects predominantly females and can occur sporadically or in patients with tuberous sclerosis complex. This review describes the recent progress in our understanding of the molecular pathogenesis of the disease and LAM cell biology. It also summarises current therapeutic approaches and the most promising areas of research for future therapeutic strategies.

Vascular smooth muscle cells ablation with endovascular nonthermal irreversible electroporation.

PURPOSE: To evaluate the effect of endovascular nonthermal irreversible electroporation (NTIRE) on blood vessels. MATERIALS AND METHODS: Specially made endovascular devices with four electrodes on top of inflatable balloons were used to apply electroporation pulses. Finite element simulations were used to characterize NTIRE protocols that would not induce thermal damage to treated tissues. Right iliac arteries of eight rabbits were treated with 90 NTIRE pulses. Angiograms were performed before and after the procedures. Arterial specimens were harvested at 7 and 35 days. Evaluation included hematoxylin and eosin, elastic von Giessen, and Masson trichrome stains. Immunohistochemistry of selected slides included smooth muscle actin (SMA), proliferating cell nuclear antigen, von Willebrand factor (VWF), and S-100 antigen. RESULTS: At 7 days, all NTIRE-treated arterial segments displayed complete, transmural ablation of vascular smooth muscle cells (VSMC). At 35 days, similar damage to VSMC was noted. In most cases, the elastic lamina remained intact, and endothelial layer regenerated. Occasional mural inflammation and cartilaginous metaplasia were noted. After 5 weeks, there was no evidence of significant VSMC proliferation, with the dominant process being wall fibrosis with regenerated endothelium. CONCLUSIONS: NTIRE can be applied in an endovascular approach. It efficiently ablates vessel wall within seconds and with no damage to extracellular structures. NTIRE has possible applications in many fields of clinical cardiology, including arterial restenosis and cardiac arrhythmias.

Compositional volumetry of non-calcified coronary plaques by multislice computed tomography: an ex vivo feasibility study.

AIMS: Non-invasive quantitative compositional analysis of coronary plaque would be a major advantage to study coronary artery disease. This study explores the application to use the Hounsfield units (HU) distribution of coronary plaques imaged by multislice computed tomography-coronary angiography (MSCT-CA). METHODS AND RESULTS: A dedicated computer-assisted method was developed to measure the HU distribution within a coronary plaque by MSCT-CA. To test the feasibility of the method, an ex vivo left anterior descending (LAD) coronary specimen, excised during autopsy, was imaged both by non-enhanced and enhanced MSCT-CA. Quantitative histology was used as a reference. To test the feasibility of the new volumetric analytic method, the MSCT-CA data were compared with volumetric histopathology. The coronary specimen, with a heterogeneously distributed plaque composition without large areas of calcification, was histologically sampled at five different locations, 5 mm apart, where at each location 15 sections were taken at 100 microm intervals, resulting in 75 individual histology sections. Tri-chrome Masson staining was used for histology quantification of three plaque/tissue components: smooth muscle cells (SMC), collagen and calcium. MSCT plaque composition was defined as "lower-HU" or "higher-HU" plaque and "calcium" based on the HU distribution. Comparison of the MSCT defined tissue components against histology showed a good relationship without significant differences. CONCLUSIONS: This ex vivo study shows the feasibility of using the Hounsfield unit distribution to perform compositional coronary plaque volumetry by MSCT-CA. The results are encouraging.

Characterization of the column and autocellular junctions that define the vasculature of gill lamellae.

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.

Second Annual Mario S. Verani, MD, Memorial Lecture: Nuclear cardiology, the next 10 years.

The nuclear cardiology of the future will be based on new clinical and biologic targets. It will be driven by modern concepts of molecular and cell biology and molecular genetics. A major effort involves detection of atherosclerosis and vascular vulnerability. Approaches include targeting proliferating smooth muscle cells, angiogenesis, vascular injury, inflammation through a variety of mechanisms, defining cell death and protease activation, and imaging gene expression. Another new clinical target involves imaging stem cells and various progenitor cells. To meet these new objectives, advanced imaging technology is required. This involves the development of micro-single photon emission computed tomography and micro-positron emission tomography systems as well as fusion technology involving radiologic computed tomography imaging together with nuclear imaging. Vascular lesion detection imaging may require intravascular detectors. The future of nuclear cardiology, based on molecular imaging, is extraordinarily exciting. The newly defined biologic targets will allow the answering of many of the key clinical questions that will dominate cardiovascular care in cardiovascular investigation over the next decade.

In vivo noninvasive identification of cell composition of intimal lesions: a combined approach with ultrasonography and immunocytochemistry.

PURPOSE: We investigated whether differences in cellular composition of the shoulder region of carotid plaque, a cell-rich, debris-free area, can be revealed with computer-driven analysis of ultrasound scans. METHODS: In 26 patients referred for carotid endarterectomy, the shoulder region of plaque eligible for surgical removal was identified with ultrasound scanning. Digital images were obtained and evaluated with a specially developed computer-driven system (Medical Image Processing [MIP]). The gray level distribution of the region of interest (ROI), along with some statistical parameters exploring the spatial distribution of pixels, such as entropy and second angular moment, were analyzed. In the specimen retrieved at surgery, the area corresponding to the ROI was selected. Cryosections were tested at immunocytochemistry with monoclonal antibodies specific to smooth muscle cells (SMCs), macrophages), and lymphocytes. Computerized image analysis was performed to quantify each cellular component of the lesion. RESULTS: Mean gray levels were related positively to the content of SMCs (r = 0.576, P =.002) and negatively to the content of macrophages (r = -0.555, P =.003). Lymphocytes did not show any correlation. Prevalence of SMCs, expressed as the ratio SMC/(SMC + macrophages), was related positively with entropy (r = 0.517, P =.007) and negatively with the second angular moment (r = -0.422, P =.032). The quartiles of gray level were useful for detecting significant differences in terms of cellular composition. CONCLUSIONS: Some cellular features of the shoulder region of plaque are associated with specific videodensitometric patterns evaluated with MIP. This approach enables in vivo noninvasive prediction and monitoring of cell composition of the shoulder region, and could be extended to study of the thickened intima.

Balloon dilation-assisted laparoscopic heller myotomy and Dor fundoplication.

Two methods are currently used in the treatment of achalasia: endoscopic balloon dilation and surgical Heller myotomy. Laparoscopy has come into use in achalasia surgery, and good outcomes have been achieved. This study included 11 patients (mean age, 30.7 years). Balloon dilation-assisted laparoscopic Heller myotomy and Dor fundoplication were performed in all patients. A 36-F orogastric tube was placed under visualization. The balloon of the tube was placed in the esophagogastric junction. After laparoscopic cardiomyotomy, the balloon was removed and Dor fundoplication was performed. The mean operative time was 90 minutes. The patients were discharged on the second and third postoperative days (mean [standard deviation], 3 +/- 0.46). One month after the operation, the patients were tested with barium swallowing, and no complications or recurrences were observed. Laparoscopic distal esophagomyotomy combined with partial fundoplication may be the surgical approach of choice in achalasia because it is safer, provides good to excellent relief of symptoms and excellent cosmetic results, involves a shorter hospital stay, and is easy to execute. Balloon dilation makes myotomy easier because it separates the muscle fibers. Placing and insufflation of the balloon become safer because the entire procedure is executed under visualization; thus, excessive dilation is avoided.