Pulmonary Tumor Thrombotic Microangiopathy in a Patient with Rapid Progressive Triple-Negative Breast Cancer.

Introduction: Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare complication of metastatic carcinoma, which occurs in patients with pulmonary arterial hypertension, and is mostly fatal. Circulating tumor cell clusters have been recognized as critical factors during breast cancer progression. Case Presentation: An 80-year-old woman with triple-negative breast cancer was admitted to our hospital with progressive dyspnea and lower back pain. Breast cancer treatment included mastectomy, neoadjuvant and adjuvant chemotherapy as well as adjuvant radiotherapy, receiving her last cycle of radiotherapy 8 days before death. At admission, D-dimers were strongly elevated and platelets were low. NT-pro-BNP was moderately elevated. A CT scan of the chest did not show pulmonary embolism but revealed interlobular septal thickening, centrilobular consolidation, and distension of the pulmonary arteries. Moreover, new skeletal and most likely lymphatic metastasis was described. Treatment with oxygen and oral glucocorticoids was initiated, assuming radiotherapy-induced pneumonitis. Due to low expression of PD-L1 and her markedly bad performance status, tumor-specific therapy was not possible, and the treatment regimen was changed to best supportive care. The patient died 8 days after admission. Autopsy revealed numerous events consistent with tumor emboli in the pulmonary vessels, suggesting PTTM. Conclusion: PTTM is a rare and mostly fatal complication in malignant breast cancer. As an early detection is difficult, further investigation is needed. Circulating tumor cluster cells may be one way to detect PTTM early and improve patients' survival.

Evaluation of a collagen-bioaggregate composite scaffold in the repair of sheep pulp tissue.

PURPOSE: This study aimed to compare the effects of the collagen-BioAggregate mixture (CBA-M) and collagen-BioAggregate composite (CBA-C) sponge as a scaffolding material on the reparative dentin formation. MATERIALS AND METHODS: CBA-C sponge (10:1 w/w) was obtained and characterized by Scanning Electron Microscopy (SEM) and Mercury Porosimetry. Cytotoxicity of the CBA-C sponge was tested by using the L929 mouse fibroblast cell line. Dental pulp stem cells (DPSCs) were isolated from the pulp tissue of sheep teeth and characterized by flow cytometry for the presence of mesenchymal stem cell marker, CD44. The osteogenic differentiation capability of isolated DPSCs was studied by Alizarin Red staining. The cells were then used to study for the compatibility of CBA-C sponge with cell proliferation and calcium phosphate deposition. The effect of CBA-C sponge and CBA-M on the induction of dentin regeneration was studied in the perforated teeth of sheep for the eight-week period. All the analyses were performed with appropriate statistical hypothesis tests. RESULTS: CBA-C sponge was found to be biocompatible for DPSCs. The DPSCs seeded on the CBA-C sponge were able to differentiate into the osteoblastic lineage and deposit calcium phosphate crystals in vitro. Reparative dentin formation was observed after the second week in the CBA-C sponge applied group. At the end of eight weeks, a complete reparative dentin structure was formed in the CBA-C sponge applied group, whereas necrotic tissue residues were observed in groups treated with the CBA-M. CONCLUSION: CBA-C sponge represents a better microenvironment for reparative dentin formation probably due to maintaining DPSCs and allowing their osteogenic differentiation and thus calcium phosphate deposition.

3D cellular alignment and biomimetic mechanical stimulation enhance human adipose-derived stem cell myogenesis.

Determination of a stem cell source with sufficient myogenic differentiation capacity that can be easily obtained in large quantities is of great importance in skeletal muscle regeneration therapies. Adipose-derived stem cells (ASCs) are readily available, can be isolated from fat tissue with high yield and possess myogenic differentiation capacity. Consequently, ASCs have high applicability in muscle regenerative therapies. However, a key challenge is their low differentiation efficiency. In this study, we have explored the potential of mimicking the natural microenvironment of the skeletal muscle tissue to enhance ASC myogenesis by inducing 3D cellular alignment and using dynamic biomimetic culture. ASCs were entrapped and 3D aligned in parallel within fibrin-based microfibers and subjected to uniaxial cyclic stretch. 3D cell alignment was shown to be necessary for achieving and maintaining the stiffness of the construct mimicking the natural tissue (12 ± 1 kPa), where acellular aligned fibers and cell-laden random fibers had stiffness values of 4 ± 1 and 5 ± 2 kPa, respectively, at the end of 21 d. The synergistic effect of 3D cell alignment and biomimetic dynamic culture was evaluated on cell proliferation, viability and the expression of muscle-specific markers (immunofluorescent staining for MyoD1, myogenin, desmin and myosin heavy chain). It was shown that the myogenic markers were only expressed on the aligned-dynamic culture samples on day 21 of dynamic culture. These results demonstrate that 3D skeletal muscle grafts can be developed using ASCs by mimicking the structural and physiological muscle microenvironment.

Development of PEI-RANK siRNA Complex Loaded PLGA Nanocapsules for the Treatment of Osteoporosis.

Osteoporosis, which is characterized by low bone mineral density and susceptibility to fracture, is caused by increased osteoclastic activity. Receptor activator of nuclear factor kappa B ligand (RANKL)/RANK signaling plays an important role in osteoclast differentiation and activation. The current treatment strategies for osteoporosis do not directly address this underlying cause and generates undesired side effects. This led to emergence of controlled delivery systems to increase drug bioavailability and efficacy specifically at the bone tissue. With better understanding of molecular pathology of bone, the use of small interfering RNA (siRNA) to inhibit translation of abnormal gene expression in cells is becoming a promising approach. In this study, we report a siRNA delivery system consisting of PEI:RANK siRNA complex entrapped in nanosized poly(lactic acid-co-glycolic acid) (PLGA) capsules intended to be used in the treatment of osteoporosis. The nanosize will enable the nanoparticles to be administered by intravenous injection. The RANK siRNA was complexed with polyethylenimine (PEI) and loaded into biodegradable PLGA nanocapsules (NCs). The PEI:RANK siRNA loaded nanocapsules significantly reduced (47%) RANK mRNA levels. The differentiation of osteoclast precursors to mature osteoclasts was significantly suppressed (∼54%). The reduction in the osteoclastic activity of the differentiated osteoclasts (55%) was found to be statistically significant. The siRNA delivery system developed in the study is planned to be tested i.v. in mouse and has the potential to be used as a novel alternative approach for the systemic treatment of osteoporosis.

Construction of a PLGA based, targeted siRNA delivery system for treatment of osteoporosis.

Osteoporosis, a systemic skeletal disorder, occurs when bone turnover balance is disrupted. With the identification of the genes involved in the pathogenesis of the disease, studies on development of new treatments has intensified. Short interfering RNA (siRNA) is used to knockdown disease related gene expressions. Targeting siRNA in vivo is challenging. The maintenance of therapeutic plasma level is hampered by clearance of siRNA from the body. Targeted systems are useful in increasing the drug concentration at the target site and decreasing side effects. Aim of the present study was to develop an injectable siRNA delivery system to protect siRNA during systemic distribution and target the siRNA to bone tissue using a thermoresponsive, genetically engineered, elastin-like recombinamer (ELR), designed to interact with the mineral component of bone. The delivery system consisted of DNAoligo as a siRNA substitute complexed with the cationic polymer, polyethyleneimine (PEI), at N/P ratio of 20. The complex was encapsulated in poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules. PLGA capsules were characterized by SEM, TEM and XPS. FTIR was used to show the preferential attachment of ELR to HAp. Encapsulation efficiency of the complex in PLGA nanocapsules was 48%. The release kinetics of the complex fits the Higuchi release kinetics.

Quantitative dynamic contrast-enhanced ultrasound for the functional evaluation of the skeletal muscle microcirculation in systemic sclerosis.

PURPOSE: The purpose was to evaluate skeletal muscle microcirculation by means of quantitative dynamic contrast-enhanced ultrasound (DCEUS) in patients with systemic sclerosis (SSc). METHODS: DCEUS imaging of the gastrocnemius muscle was performed with phospholipid-stabilized microbubbles filled with sulfur hexafluoride in 12 patients with SSc and 12 healthy volunteers. The fitted time intensity curves (TIC) during the first 3 minutes after administration of the contrast agent bolus were analysed. Time course parameters of the TIC were compared between patients and healthy volunteers. RESULTS: Peak enhancement, wash-in area under the curve and was-out area under the curve was decreased in the patient group versus healthy volunteers (168 versus 248 AU p = 0.291; 2193 versus 3314 p = 0.198; 4948 AU x sec versus 8948 AU x sec p = 0.037). In the SSc patients the mean transit time and wash-in perfusion index were numerically, but not statistically lower than in the healthy volunteers, but rise and fall time were similar. CONCLUSION: On the microvascular level in SSc patients versus their healthy counterpart key parameters related to blood volume were decreased and perfusion parameters showed a slight diminishment in the patient population. These results suggest a component of impaired skeletal muscle microcirculation in SSc patients.

Assessment of cutaneous microcirculation in unaffected skin regions by transcutaneous oxygen saturation monitoring and Laser Doppler flowmetry in systemic sclerosis.

We assessed the cutaneous microcirculatory reactivity of a clinically unaffected skin region in patients with systemic sclerosis (SSc) compared to healthy controls by measuring transcutaneous oxygen saturation (TcPO2) and Laser Doppler flowmetry (LDF).Twelve consecutive patients with SSc and twelve healthy controls were subjected to TcPO2 monitoring and LDF during cuff-induced ischemia and reactive hyperemia in order to measure the skin oxygen tension and the microcirculatory blood flow. Mean minimal and maximal values of oxygen tension and blood flow, time to peak (TTP), and declining slopes after peaking (slope) were compared between patients with SSc and controls.Compared to the controls, TcPO2 values in SSc were similar during ischemia and diminished during reactive hyperemia, with longer TTP, and a slower return to baseline (-60% vs. -58% , p = 1.000, +76% vs. +210% , p = 0.047, 137 s vs. 108 s, p = 0.028, -0.009% /s vs. -0.019% /s, p = 0.021, respectively). LDF values, however, did not differ significantly between patients with SSc and controls.Unaffected skin regions of SSc patients showed a significantly diminished postischemic vasodilatory reactivity when assessed by TcPO2 monitoring, but not by LDF, indicating that vasculopathy may represent an early mechanism in the onset of skin sclerosis. TcPO2 measurement may help to detect changes in the microcirculation in SSc with no skin affection.

Assessing the end-organ in peripheral arterial occlusive disease-from contrast-enhanced ultrasound to blood-oxygen-level-dependent MR imaging.

Peripheral arterial occlusive disease (PAOD) is a result of atherosclerotic disease which is currently the leading cause of morbidity and mortality in the western world. Patients with PAOD may present with intermittent claudication or symptoms related to critical limb ischemia. PAOD is associated with increased mortality rates. Stenoses and occlusions are usually detected by macrovascular imaging, including ultrasound and cross-sectional methods. From a pathophysiological view these stenoses and occlusions are affecting the microperfusion in the functional end-organs, such as the skin and skeletal muscle. In the clinical arena new imaging technologies enable the evaluation of the microvasculature. Two technologies currently under investigation for this purpose on the end-organ level in PAOD patients are contrast-enhanced ultrasound (CEUS) and blood-oxygen-level-dependent (BOLD) MR imaging (MRI). The following article is providing an overview about these evolving techniques with a specific focus on skeletal muscle microvasculature imaging in PAOD patients.

Correlation of skeletal muscle blood oxygenation level-dependent MRI and skin laser Doppler flowmetry in patients with systemic sclerosis.

PURPOSE: To investigate the origin of skeletal muscle BOLD MRI alterations in patients with systemic sclerosis (SSc) by correlating BOLD MRI T2* signal of calf muscles with microcirculatory blood flow of calf skin measured by laser Doppler flowmetry (LDF). MATERIALS AND METHODS: BOLD MRI (3T) and LDF measurements were performed in 12 consecutive SSc patients (6 women, 6 men; mean age 54.0 ± 10.0 years) and 12 healthy volunteers (4 men, 8 women; mean age 44.7 ± 13.1 years). For both modalities, the same cuff compression paradigm at mid-thigh level was used. LDF datasets were acquired using a PeriScan PIM II Imager (Perimed AB, Stockholm, Sweden) at the upper calf corresponding to the level of MR imaging. Cross-correlations of BOLD and LDF signal intensity changes depending on time lags between both time series were calculated. RESULTS: Maximal cross-correlations of BOLD T2* and LDF measurements were calculated as 0.93 (healthy volunteers) and 0.94 (SSc patients) for a BOLD time lag of approximately 10 s. Key parameter analysis suggested that in contrast to hyperemic BOLD signal loss at maximum value in SSc patients, ischemic T2* decrease cannot be explained by differences of tissue perfusion. CONCLUSION: Skeletal muscle BOLD T2* signal in SSc patients is closely correlated with changes of microperfusion as detected by LDF.

Correlation of muscle BOLD MRI with transcutaneous oxygen pressure for assessing microcirculation in patients with systemic sclerosis.

PURPOSE: To prospectively compare calf muscle BOLD MRI with transcutaneous oxygen pressure (TcPO2 ) measurement in patients with systemic sclerosis (SSc) and healthy volunteers and thereby get insight into the pathogenesis of vasculopathy in this connective tissue disorder. MATERIALS AND METHODS: Twelve patients with SSc (6 women and 6 men, mean age 53.5 ± 10.0 years) and 12 healthy volunteers (4 men and 8 women, mean age 47 ± 12.1 years) were examined using muscle BOLD MRI and TcPO2. A cuff compression at mid-thigh level was performed to provoke ischemia and reactive hyperemia. BOLD measurements were acquired on a 3 Tesla whole body-scanner in the upper calf region using a multi-echo EPI-sequence with four echo-times (TE: 9/20/31/42 ms) and a repetition time of 2 s. Empirical cross-correlation analysis depending on time lags between BOLD- and TcPO2-measurements was performed. RESULTS: Maximal cross-correlation of BOLD T2*- and TcPO2-measurements was calculated as 0.93 (healthy volunteers) and 0.90 (SSc patients) for a time lag of approximately 40 s. Both modalities showed substantial differences regarding time course parameters between the SSc patients and healthy volunteers. CONCLUSION: Skeletal muscle BOLD MRI correlated very well with TcPO2 . T2* changes seem to reflect reoxygenation deficits in deeper muscle tissue of SSc patients.

Integration of the computational fluid dynamics technique with MRI in aortic dissections.

Short-term and long-term prognosis and their determining factors of Type III/Stanford B aortic dissections (TB-AD), which separate the aorta distal at the origin of the subclavian artery into a true lumen and false lumen, have been elusive: One quarter of patients thought to be treated successfully, either by medical or by surgical means, do not survive 3 years. Unfavorable hemodynamic conditions are believed to lead to false lumen pressure increases and complications. A better characterization of TB-AD hemodynamics may therefore impact therapeutic decision making and improve outcome. The large variations in TB-AD morphology and hemodynamics favor a patient-specific approach. Magnetic resonance imaging with its capability to provide high-resolution structural images of the lumen and aortic wall and also to quantify aortic flow and kinetics of an exogenous tracer is a promising clinical modality for developing a deeper understanding of TB-AD hemodynamics in an individual patient. With the information obtained with magnetic resonance imaging, computational fluid dynamics simulations can be performed to augment the image information. Here, an overview of the interplay of magnetic resonance imaging and computational fluid dynamics techniques is given illustrating the synergy of these two approaches toward a comprehensive morphological and hemodynamic characterization of TB-AD.

ECG-triggered non-enhanced MR angiography of peripheral arteries in comparison to DSA in patients with peripheral artery occlusive disease.

OBJECT: The purpose of this study was to evaluate peripheral non-enhanced-MRA (NE-MRA) acquired with a 3D Turbo Spin Echo sequence with electrocardiographt (ECG) triggering in comparison to Digital Subtraction Angiography (DSA) as the gold standard in symptomatic peripheral artery occlusive disease (PAOD) patients. MATERIALS AND METHODS: This IRB approved prospective study included 23 PAOD patients from whom three patients had to be excluded. The remaining 20 subjects were included in the analysis (15 male; mean age 62.4 ± 15.3 years). The patients first underwent DSA followed by NE-MRA on a 1.5-T whole body scanner within 24 h after the DSA study. A NATIVE (Non-contrast Angiography of the Arteries and Veins) SPACE (Sampling Perfection with Application Optimized Contrast by using different flip angle Evolution) sequence at four levels (pelvis, upper leg, knee region and lower leg) was acquired. For evaluation purposes, subtracted standardized MIP (maximum intensity projection) images were generated from the NE-MRA data sets. Qualitative assessment of NE-MRA images in reference to the corresponding DSA images, as well as blinded stenosis grading of preselected segments in NE-MRA images were performed by two experienced readers. Image quality in 95 corresponding arterial segments was rated from 1 (good) to 4 (inadequate) directly comparing the NE-MRA with the corresponding DSA segment as the gold standard. Blinded stenosis grading consisted of 66 preselected stenoses rated from 1 (<10 %) to 4 (>90 %) in NE-MRA which were compared to the grade in the corresponding DSA. RESULTS: The mean image quality of NE-MRA in comparison to DSA was 2.7 ± 1.1 (reader 1) and 3.0 ± 1.0 (reader 2). The kappa value indicating interobserver agreement was 0.34; readers 1 and 2 rated the image quality as good in 21 % and 3 %, sufficient in 19 % and 41 %, limited in 29 % and 14 % and inadequate in 31 % and 42 %, respectively. Stenosis graduation revealed significantly higher grades in NE-MRA (reader 1: 3.0 ± 0.7, p < 0.001 and reader 2: 3.1 + 0.8, p < 0.001) compared to DSA (mean value DSA 2.7 ± 0.8). The kappa value indicating interobserver agreement concerning stenosis grading was 0.59. CONCLUSION: NE-MRA revealed a relatively high number of inadequate quality segments. This is in line with recently published comparable studies of the similar SPACE NE-MRA techniques. Further advance of NE-MRA techniques remains desirable for patients with PAOD.

Impaired skeletal muscle microcirculation in systemic sclerosis.

INTRODUCTION: Muscle symptoms in systemic sclerosis (SSc) may originate from altered skeletal muscle microcirculation, which can be investigated by means of blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI). METHODS: After ethics committee approval and written consent, 11 consecutive SSc patients (5 men, mean age 52.6 years, mean SSc disease duration 5.4 years) and 12 healthy volunteers (4 men, mean age 45.1 years) were included. Subjects with peripheral arterial occlusive disease were excluded. BOLD MRI was performed on calf muscles during cuff-induced ischemia and reactive hyperemia, using a 3-T whole-body scanner (Verio, Siemens, Erlangen, Germany) and fat-suppressed single-short multi-echo echo planar imaging (EPI) with four different effective echo times. Muscle BOLD signal time courses were obtained for gastrocnemius and soleus muscles: minimal hemoglobin oxygen saturation (T2*min) and maximal T2* values (T2*max), time to T2* peak (TTP), and slopes of oxygen normalization after T2* peaking. RESULTS: The vast majority of SSc patients lacked skeletal muscle atrophy, weakness or serum creatine kinase elevation. Nevertheless, more intense oxygen desaturation during ischemia was observed in calf muscles of SSc patients (mean T2*min -15.0%), compared with controls (-9.1%, P = 0.02). SSc patients also had impaired oxygenation during hyperemia (median T2*max 9.2% vs. 20.1%, respectively, P = 0.007). The slope of muscle oxygen normalization was significantly less steep and prolonged (TTP) in SSc patients (P<0.001 for both). Similar differences were found at a separate analysis of gastrocnemius and soleus muscles, with most pronounced impairment in the gastrocnemius. CONCLUSIONS: BOLD MRI demonstrates a significant impairment of skeletal muscle microcirculation in SSc.

Skeletal muscle BOLD MRI: from underlying physiological concepts to its usefulness in clinical conditions.

Blood oxygenation-level dependent (BOLD) MRI has gained particular attention in functional brain imaging studies, where it can be used to localize areas of brain activation with high temporal resolution. To a higher degree than in the brain, skeletal muscles show extensive but transient alterations of blood flow between resting and activation state. Thus, there has been interest in the application of the BOLD effect in studying the physiology of skeletal muscles (healthy and diseased) and its possible application to clinical practice. This review outlines the potential of skeletal muscle BOLD MRI as a diagnostic tool for the evaluation of physiological and pathological alterations in the peripheral limb perfusion, such as in peripheral arterial occlusive disease. Moreover, current knowledge is summarized regarding the complex mechanisms eliciting BOLD effect in skeletal muscle. We describe technical fundaments of the procedure that should be taken into account when performing skeletal muscle BOLD MRI, including the most often applied paradigms to provoke BOLD signal changes and key parameters of the resulting time courses. Possible confounding effects in muscle BOLD imaging studies, like age, muscle fiber type, training state, and drug effects are also reviewed in detail.

Clinical implications of skeletal muscle blood-oxygenation-level-dependent (BOLD) MRI.

Blood-oxygenation-level-dependent (BOLD) contrast in magnetic resonance (MR) imaging of skeletal muscle mainly depends on changes of oxygen saturation in the microcirculation. In recent years, an increasing number of studies have evaluated the clinical relevance of skeletal muscle BOLD MR imaging in vascular diseases, such as peripheral arterial occlusive disease, diabetes mellitus, and chronic compartment syndrome. BOLD imaging combines the advantages of MR imaging, i.e., high spatial resolution, no exposure to ionizing radiation, with functional information of local microvascular perfusion. Due to intrinsic contrast provoked via changes in hemoglobin oxygen saturation, it is a safe and easy applicable procedure on standard whole-body MR devices. Therefore, BOLD MR imaging of skeletal muscle is a potential new diagnostic tool in the clinical evaluation of vascular, inflammatory, and muscular pathologies. Our review focuses on the current evidence concerning the use of BOLD MR imaging of skeletal muscle under pathological conditions and highlights ways for future clinical and scientific applications.

Associated vascular lesions in patients with spontaneous coronary artery dissection.

AIM: To identify vascular abnormalities in patients presenting with spontaneous coronary artery dissection (SCAD). METHODS: We performed a whole-body MR angiography and a duplex sonography of the renal and carotid arteries in 12 patients (9 women, 3 men) with SCAD to identify vascular abnormalities. RESULTS: MR angiography revealed abnormalities of the renal arteries in 3/12 patients (25%). All 3 patients were women, 2 presented with changes suggesting fibromuscular dysplasia (FMD), 1 had a spontaneous renal artery dissection. No other vascular abnormalities were identified in any of the patients. Duplex sonography confirmed MR findings and showed non-significant renal artery stenoses in both patients with FMD. CONCLUSIONS: Abnormalities of the renal arteries were found in 3/12 (25%) of the patients with SCAD. No other vascular abnormalities were identified. Additional diagnostic tests of the renal arteries such as renal artery angiography or duplex sonography may be considered in patients presenting with SCAD.

Blood oxygenation level-dependent (BOLD) MRI of human skeletal muscle at 1.5 and 3 T.

PURPOSE: To evaluate the dependence of skeletal muscle blood oxygenation level-dependent (BOLD) effect and time course characteristics on magnetic field strength in healthy volunteers using an ischemia/reactive hyperemia paradigm. MATERIALS AND METHODS: Two consecutive skeletal muscle BOLD magnetic resonance imaging (MRI) measurements in eight healthy volunteers were performed on 1.5 T and 3.0 T whole-body MRI scanners. For both measurements a fat-saturated multi-shot multiecho gradient-echo EPI sequence was applied. Temporary vascular occlusion was induced by suprasystolic cuff compression of the thigh. T2 time courses were obtained from two different calf muscles and characterized by typical curve parameters. Ischemia- and hyperemia-induced changes in R2 (ΔR2) were calculated for both muscles in each volunteer at the two field strengths. RESULTS: Skeletal muscle BOLD changes are dependent on magnetic field strength as the ratio ΔR2(3.0 T)/ΔR2(1.5 T) was found to range between 1.6 and 2.2. Regarding time course characteristics, significantly higher relative T2 changes were found in both muscles at 3.0 T. CONCLUSION: The present study shows an approximately linear field strength dependence of ΔR2 in the skeletal muscle in response to ischemia and reactive hyperemia. Using higher magnetic fields is advisable for future BOLD imaging studies of peripheral limb pathologies.