Movat Pentachrome Research Articles

Development and Evaluation of a Disease Large Animal Model for Preclinical Assessment of Renal Denervation Therapies.

Simple SummaryHigh blood pressure (arterial hypertension) is a major public health problem as far as it has a high prevalence in the adult population and it entails significant cardiovascular and cerebrovascular risks. New ways of treating hypertension include catheter-based renal denervation, i.e., modulation of the autonomic nervous system that has a crucial role in the regulation of blood pressure. We have designed a hypertensive animal model to test the efficacy and safety of this kind of treatment. After the induction of mineralocorticoid mediated hypertension in minipigs, we have analyzed the durability of hypertensive state, the response to drugs and the effects of renal denervation obtained with a radiofrequency catheter (Symplicity®, Medtronic, Santa Rosa, CA, USA). The attrition rate was high, mainly caused by infections in this instrumentalized animal model. The effect of renal denervation was confirmed by pathology (nerve damage), biochemical parameters and blood pressure measurements. The conclusion is that this model could be used in the assessment of different therapies aimed to modulate the influence of the autonomic nervous system on hypertension if the high mortality rate is reduced with a less invasive monitorization system.New-generation catheters-based renal denervation (RDN) is under investigation for the treatment of uncontrolled hypertension (HTN). We assessed the feasibility of a large animal model of HTN to accommodate the human RDN devices. Ten minipigs were instrumented to measure blood pressure (BP) in an awake-state. HTN was induced with subcutaneous 11-deoxycorticosterone (DOCA, 100 mg/kg) implants. Five months after, the surviving animals underwent RDN with the Symplicity® system. Norepinephrine (NE) renal gradients were determined before and 1 month after RDN. Renal arteries were processed for histological (hematoxylin-eosin, Movat pentachrome) and immunohistochemical (S100, tyrosine-hydroxylase) analyses. BP significantly rose after DOCA implants. Six animals died prematurely, mainly from infectious causes. The surviving animals showed stable BP levels after 5 months. One month after RDN, nerve damage was showed in three animals, with impedance drop >10%, NE gradient drop and reduction in BP. The fourth animal showed no nerve damage, impedance drop <10%, NE gradient increase and no change in BP. In conclusion, the minipig model of DOCA-induced HTN is feasible, showing durable effects. High mortality should be addressed in next iterations of this model. RDN may partially offset the DOCA-induced HTN. Impedance drop and NE renal gradient could be markers of RDN success.

Size-Optimized Microspace Culture Facilitates Differentiation of Mouse Induced Pluripotent Stem Cells into Osteoid-Rich Bone Constructs.

Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560 μm (Elp400), 500/700 μm (Elp500), and 900/700 μm (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2, Osterix, Collagen 1a1, Osteocalcin, Bone sialoprotein, and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat's pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.

Sub-classification of non-inflammatory and inflammatory surgical aortic aneurysms and the association of histological characteristics with potential risk factors.

The present study aimed to analyze the histological characteristics of surgical thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) specimens on the basis of the most recent consensus documents on non-inflammatory and inflammatory lesions. The current study also aimed to establish an association with various risk factors. Aortic wall specimens were collected from 52 patients (38 men and 14 women; age, 19–80 years) undergoing surgery for aortic dilatation at The Cardiovascular Disease Institute (Iasi, Romania). For histological evaluation, the aortic specimens (39 TAAs and 13 AAAs) were stained with hematoxylin-eosin, Van Giessen, alcian blue and Movat pentachrome. The specimens were evaluated and graded according to the severity of histopathological conditions: Fragmentation of elastic fibers, medial mucoid accumulation, smooth muscle cell loss and medial fibrosis. The severity of atherosclerotic lesions in surgically resected segments of the aorta were graded as follows: i) mild=1; ii) moderate=2; and iii) severe=3. The risk factors associated with TAA were the male sex (80%), smoking (56%), hypertension (33%) and bicuspid aortic valve (13%). Advanced age (70 years), male sex (69%) and smoking (54%) were determined to be the risk factors of AAA. The histopathological abnormalities included medial degeneration (MD) (82%), atherosclerosis (ATS) (42%) and aortitis (10%). MD was the leading histopathological diagnosis in TAA and the severity of lesions were graded as follows: Mild (8% of cases), moderate (44% of cases) and severe (31% of cases). Severe atherosclerotic lesions were identified in AAA (100% of cases). In the present study, medial degenerative aortic lesions (1, mild; 2, moderate; and 3, severe) significantly correlated with advanced age (>65 years; r=−0.39; P<0.01) and male sex (r=0.27; P<0.05). Significant correlations were also identified between atherosclerotic aortic lesions (1, mild; 2, moderate; and 3, severe) and advanced age (>65 years) (r=−0.40, P<0.01) or smoking (r=−0.29; P<0.05). Advanced age, male sex and smoking were determined to be the main risk factors for the development of degenerative aortic aneurysms.

Development of a novel histological and histomorphometric evaluation protocol for a standardized description of the mid-palatal suture - An ex vivo study.

The hard palate and mid-palatal suture are highly important for orthodontic treatment. In cases of transverse maxillary deficiency, palatal expansion is the treatment of choice. As nowadays a growing number of adult patients receive orthodontic treatment, an understanding of suture development throughout life is important to derive tailored orthodontic treatment techniques for each age group. Histological, histochemical and immunohistochemical stains (haematoxylin & eosin, Azan, Movat pentachrome, Masson-Goldner trichrome, Sirius Red, CD 31, osteopontin and TRAP) and histomorphometric analyses were re-established to detect the structural conditions of the mid-palatal suture in human cadavers of three different age groups (20-39, 40-59 and 60-80years). The mid-palatal suture of the selected age groups (total of n=12; n=4 in every group m=f) exhibited marked differences in sutural morphology and metabolism. A wide, interdigitated and well-vascularized suture was observed in younger specimens compared with straighter and smaller sutures with fewer vessels and lower bone density in the 60-80year group. The fibre composition within the sutural gap differed between the three age groups. Delicate fibres were found in the 20-39year group, and a tightly interwoven 3D fibre-network was observed in the 40-59year group. Atrophy primarily characterized the fibres in the 60-80 year group. This evidence demonstrates differences between the evaluated groups. These results suggest that the staining methods used are suitable for the description and evaluation of the morphology and metabolism of mid-palatal sutures. Further investigation is necessary to provide an in-depth description of sutural maturation over a lifetime.

Subclinical Endocarditis Might be a Hidden Trigger of Early Prosthetic Valve Calcification: A Histological Study.

Despite various improvements in valve prosthetics, early valve deterioration still occurs, leading to prosthetic failure. Studying the early phase of this deterioration is quite difficult, as the prosthesis to be examined is almost always explanted only after extensive deterioration. The objective of this research is to study the pathology of early valve deterioration in an early stage in order to reveal the possible trigger of the process. Three cusps of the same type of bovine pericardium valve prosthesis underwent comparative examination. Two cusps (cusps 1 and 2) were retrieved from a valve prosthesis explanted three months post-implantation, and the third cusp was from a non-implanted valve prosthesis and used as a reference cusp (ref. cusp). The examination included macroscopic examination, Non-linear Optical Microscopy using a multiphoton microscope, and histological examination with staining, using Hematoxylin and Eosin, Movat Pentachrome stain, Von-Kossa stain, and Alizirin-Red stain. Parallel sections were decalcified using Osteosoft® solution prior to Von-Kossa and Alizirin-Red staining to exclude false positive results. Macroscopically, cusp 1 showed early deterioration, and cusp 2 showed endocarditic vegetations. Histologically, cusp 1 showed calcifications in acellular deposits on the surface of the cusp, with pathological signs of subacute/healed endocarditis and intact cusp tissue. The examination did not show calcifications of the cellular remnants within the valve tissue. Cusp 2 showed florid endocarditis, with microscopic destruction of the valve tissue. Early prosthetic valve deterioration can exist as early as three months post-implantation. Subacute or subclinical endocarditis can be the cause for early valve calcification and deterioration.

Stromal heterogeneity in pancreatic cancer and chronic pancreatitis

Background/objectivesAn abundant stromal reaction is a hallmark of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). The cells mainly responsible for the stromal reaction are activated pancreatic stellate cells (PSCs). Despite their crucial role, PSCs are not well characterized. PSCs share characteristics with the better-known hepatic stellate cells (HSCs). The aim of this study was a detailed analysis of PSCs in PDAC and CP. MethodsWhole-slide specimens of CP (n = 12) and PDAC (n = 10) were studied by histochemistry and immunohistochemistry. The stroma was evaluated using Movat's pentachrome stain. PSCs were tested by immunohistochemistry for PSC markers (α-SMA, CD34, desmin, NGFR, SPARC and tenascin C) and HSC markers (α-crystallin B, CD56, NGF, NT-3, synaptophysin and TrkC). Alpha-SMA, tenascin C, SPARC and NT-3 staining were verified on tissue micro arrays (TMAs) from a well-characterized cohort of 223 PDAC patients. PSCs isolated from human PDAC and CP tissue samples as well as HSCs were evaluated by immunofluorescence. ResultsWhile the stroma of CP cases was characterized by a collagen-rich fibrosis, PDAC stroma displayed higher mucin content (p = 0.0002). PSCs showed variable expression of tested markers. In PDAC samples, staining of most markers was found around tumor complexes, while CP samples showed a greater variety of localizations. Alpha-SMA staining correlated with collagen-rich fibrosis (p = 0.012), while NT-3 staining correlated with mucin-rich stroma (p = 0.008). A peritumoral staining was confirmed for α-SMA, tenascin C, SPARC and NT-3 in the PDAC TMA cohort (n = 223). In a subgroup of patients with pancreatic head tumors and UICC 2009 IIB (n = 144), α-SMA staining intensity was a prognostic factor for overall survival at uni- and multivariate analysis (p = 0.036 and p = 0.002). ConclusionsThe close similarities between PSCs and HSCs were confirmed. Heterogeneous expression patterns of the tested markers might reflect different levels of activation or differentiation, or even multiple subpopulations of PSCs. Survival analysis suggests an impact of stromal composition on survival.

Histology and 3D Modeling of the Connective Tissue Skeleton in the Trunk of a Lamprey (Petromyzon marinus)

Agnathan lampreys play a key role in the reconstruction of the evolutionary history of vertebrates and, more specifically, in the analysis of the biomechanics underlying piscine undulatory locomotion. However, the morphology of the fibrous skeletal framework for their trunk musculature is still incompletely understood, mainly because of the complex spatial configuration of the segmentally arranged myosepta and muscle fibers within the myomeres. To contribute to a better understanding of how contractile muscular forces are transmitted via the fibrous skeletal framework of the trunk, the architecture and composition of the connective tissue structures (dermis, dorsal longitudinal ligament, hypodermis, myosepta, perimysial lamellae, notochord, fasciae) and their relationship to the muscle fibers of the myomeres were analyzed through histological techniques (in addition to MRI and microdissection described elsewhere) and 3D modeling based on a 3D reconstruction of serial histological sections treated with special stains (e.g., Movat Pentachrome, Ladewig Trichrome). These stains, however, did not differentiate tissue types as expected, probably because lamprey tissue fiber types differ biochemically from mammalian ones, but they discriminate a wide range of fiber types. As to the distribution of connective tissue fibers, the dermis comprises about 25 layers of helically arranged collagen fibers with the most superficial layers anchoring to the basement membrane of the epidermis and the deepest layers connecting to fibers of the hypodermis, which is separated by the pigment layer from the dermis. Radially arranged collagen fibers interconnect the basement membrane and hypodermis. Also within the hypodermis, the dorsal longitudinal ligament collects and interbraids collagen fibers from the dermis, hypodermis, and dorsal vertical septum, and, ultimately, the myosepta. The latter comprise tendon fibers connecting the roughly longitudinally oriented muscle fibers of adjacent myomeres and are traversed by thick collagen fiber bundles that run from the dermis to the central periaxial and perivisceral fasciae, as well as by thin reticulated fibers that support intramyoseptal nerves and blood vessels. The muscle blocks of the myomeres are separated by radially arranged perimysial lamellae running from the hypodermis to the periaxial and perivisceral fasciae. During undulations, contracting muscle fibers between the myosepta shorten and expand radially. This expansion is resisted by the radially arranged collagen fibers within the myosepta and perimysial lamellae. As a result, the internal pressure and, hence, the turgidity of the trunk is raised. A detailed understanding of the connective tissue fiber configuration in lampreys provides the necessary basis for building dynamic 3D models of the biomechanics involved in locomotion and other behaviors.Support or Funding InformationGrant‐In‐Aid of Research from Sigma Xi, the Scientific Research Society, to Bradley M. Wood; LSU Foundation support to Dominique G. HombergerThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pathology of stent implantation in internal mammary artery.

The internal mammary artery (IMA) is the most durable conduit for bypassing the left anterior descending (LAD) coronary artery in patients undergoing coronary artery bypass graft surgery (CABG). However, little is known about how the IMA reacts histologically to stent implantation. From CVPath stent registry (1048 lesions, 614 cases), we obtained 4 stent lesions (2 bare metal stents, 2 drug-eluting stents) involving IMA grafts. The mean age of our patients was 63years and the duration of stent implantation in the IMA ranged from 5days to 5years. Stented arteries were dissected from hearts and embedded in plastic, segmented at 3mm intervals, sectioned at 4-6microns and stained with H&E and Movat pentachrome stains. Histological observations were performed. Majority of stents (3 of 4) were implanted in anastomosis between IMA and LAD while 1 stent was implanted in IMA body. One stent with duration of 5days showed stent thrombosis while others were all patent with fully coverage by varying degrees of neointima. Foamy macrophage, lipid pool and calcification in neointima were observed in 1 stent with duration of 5years but it was limited only to the distal LAD part within the stented segment. Overall, in this small pathologic series, the majorities of stents were implanted in IMA-to-LAD anastomosis site and demonstrated acceptable pathologic responses.

Tracheal repair with acellular human amniotic membrane in a rabbit model.

Surgical correction of tracheal stenosis is still a complex and challenging procedure. Acellular human amniotic membranes (AHAM) represent a promising biomaterial source for tissue regeneration. The aim of this study was to evaluate whether AHAM grafts improve tissue regeneration of the trachea in a rabbit model of tracheostomy. Twenty rabbits were randomized into 2 groups. Animals in the control group underwent surgical tracheostomy only, and animals in the AHAM group underwent surgical tracheostomy and received an AHAM graft that covered the defect site. We examined tissues at the site of tracheostomy 60days after surgery by histological analysis with haematoxylin and eosin, Movat's pentachrome stain and immunohistochemistry by analysis with antiaggrecan antibodies. The average perimeter and area of the defect 60days after surgery were smaller in animals in the control group than in the AHAM group (p=.011 and p=.011, respectively). Histological analysis of AHAM group revealed neovascularization, islands of immature cartilage, pseudostratified ciliated epithelium. and connective tissue at the site of AHAM engraftment, whereas only pseudostratified ciliated epithelium and connective tissue were observed at the defect site in tissues of animals in the control group. Regeneration of islands of immature cartilage tissue with hyaline pattern and pseudostratified ciliated epithelium were confirmed by immunohistochemistry analysis. These results indicate that AHAM engraftment could facilitate neovascularization and regeneration of immature cartilage in a model of tracheal injury. Its use may lower the risk of post-operative complications including stenosis of trachea.

Relationship between ADAMTS4 and carotid atherosclerotic plaque vulnerability in humans

BackgroundRupture of atherosclerotic plaques and the resulting thrombosis are vital causes of clinical ischemic events. Recent studies have shown that ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) is a pathogenic factor of plaque vulnerability in mice. However, the relationship between ADAMTS4 and carotid atherosclerotic vulnerable plaques in humans remains unclear. MethodsForty-eight carotid atherosclerotic plaque specimens were obtained from 48 carotid artery stenosis inpatients undergoing carotid endarterectomy. We performed hematoxylin and eosin and Movat pentachrome staining for histologic characteristics; immunohistochemical staining for ADAMTS4, versican, and macrophages; and serologic tests for ADAMTS4. Patients were divided into stable and vulnerable groups on the basis of histologic characterization according to the classification criteria of the American Heart Association. Comparison between the groups was carried out using SPSS 17.0 (SPSS Inc, Chicago, Ill). ResultsExpression of ADAMTS4 in the plaque and its serum concentration were significantly higher in the vulnerable group compared with the stable one (P = .004 and P = .021, respectively), whereas the expression of versican was lower in the vulnerable group than in the stable group (P = .015). Univariate analysis revealed that the incidence of symptomatic cerebral ischemic events and ADAMTS4 serum levels were statistically higher in the vulnerable group compared with the stable group (P = .021 and P = .029, respectively). Multivariate analysis showed that ADAMTS4 was an independent risk factor (odds ratio, 1.14; P = .038). ConclusionsOur study revealed that ADAMTS4 expression was upregulated during carotid atherosclerotic plaque development. Serum levels of ADAMTS4 were associated with increased plaque vulnerability in both symptomatic and asymptomatic patients with carotid artery stenosis. ADAMTS4 may be a potential biomarker for plaque vulnerability.

Bone replaces articular cartilage in the rat knee joint after prolonged immobilization

BackgroundLost joint range of motion (ROM) is common in chronic osteoarthritis, alters regional weight-bearing across the articular surfaces, and contributes to loss of cartilage and bone alterations. Limited data exist on the regional effects on joints subjected to chronic losses of ROM. ObjectiveTo characterize the regional replacement by bone as part of articular cartilage degeneration after prolonged immobilization. MethodsEleven rat knees were rigidly-immobilized in flexion for 32weeks with contralateral and sham-operated (n=6) knees as controls. Sagittal medial tibial epiphysis histological sections assessed the anterior (non-weight-bearing), middle and posterior (both weight-bearing) regions. We quantified the distribution of collagen I, collagen II, cartilage thickness, glycosaminoglycan (GAG) staining, Mankin scoring, and subchondral bone plate cross-sectional area. Using immunohistochemistry (IHC), we visualized blood vessels, osteoblasts, and mesenchymal stem cells (MSCs). ResultsImmobilized cartilage had increased collagen I content in the anterior tibial region with picrosirius red staining (immobilized=61±20%; contralateral=43±12%, p=0.033; sham=20±10%, p=0.028) and collagen I IHC (immobilized=40±10%; contralateral=11±4%, p=0.003; sham=5±3%, p=0.043). Articular cartilage was thinner anteriorly (18±30μm) in immobilized knees versus contralateral (124±40μm, p<0.001) and sham (125±43μm, p=0.043). GAG staining covered 2±4% of the anterior articular area in immobilized knees versus 28±12% contralaterally (p=0.003) and 26±7% in sham (p=0.043). Mankin scores in immobilized knees were 4.7±1.7 versus 0.2±0.4 and 0±0 for contralateral and sham (p=0.003, p=0.042), respectively. The trabecular bone plate area of anterior and posterior regions showed relative loss of cross-sectional area in immobilized knees compared to controls (immobilized/contralateral area ratios of 0.67 and 0.46 respectively, both p=0.003), while the area in the middle region was preserved. Movat's pentachrome stain and CD31 staining showed chondral vascular ingrowth from subchondral bone. Osteocalcin and CD90 MSC staining were decreased in immobilized knees versus contralateral (p=0.003, p=0.036 respectively). ConclusionsBony replacement characterizes articular cartilage degeneration of knees immobilized for 32weeks in the anterior, non-weight bearing region of the tibia. Replacement of cartilage by bone may have been mediated by chondral vascularization, suggesting irreversible changes. These findings stress the importance of weight-bearing and joint motion to maintain cartilage structure.

Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-mediated PCSK9 and partial carotid ligation

Studying the role of a particular gene in atherosclerosis typically requires a time-consuming and often difficult process of generating double knockouts or transgenics on ApoE−/− or LDL receptor (LDLR)−/− background. Recently, it was reported that adeno-associated-virus-8 (AAV8)-mediated overexpression of PCSK9 (AAV8-PCSK9) rapidly induced hyperlipidemia. However, using this method in C57BL6 wild-type (C57) mice, it took ~3 months to develop atherosclerosis. Our partial carotid ligation model is used to rapidly develop atherosclerosis by inducing disturbed flow in the left common carotid artery within 2 weeks in ApoE−/− or LDLR−/− mice. Here, we combined these two approaches to develop an accelerated model of atherosclerosis in C57 mice. C57 mice were injected with AAV9-PCSK9 or AAV9-luciferase (control) and high-fat diet was initiated. A week later, partial ligation was performed. Compared to the control, AAV-PCSK9 led to elevated serum PCSK9, hypercholesterolemia, and rapid atherosclerosis development within 3 weeks as determined by gross plaque imaging, and staining with Oil-Red-O, Movat's pentachrome, and CD45 antibody. These plaque lesions were comparable to the atherosclerotic lesions that have been previously observed in ApoE−/− or LDLR−/− mice that were subjected to partial carotid ligation and high-fat diet. Next, we tested whether our method can be utilized to rapidly determine the role of a particular gene in atherosclerosis. Using eNOS−/− and NOX1−/y mice on C57 background, we found that the eNOS−/− mice developed more advanced lesions, while the NOX1−/y mice developed less atherosclerotic lesions as compared to the C57 controls. These results are consistent with the previous findings using double knockouts (eNOS−/−_ApoE−/− and NOX1−/y_ApoE−/−). AAV9-PCSK9 injection followed by partial carotid ligation is an effective and time-saving approach to rapidly induce atherosclerosis. This accelerated model is well-suited to quickly determine the role of gene(s) interest without generating double or triple knockouts.

A small reduction in control tissue size yields a significant reduction in digital image storage cost

As digital pathology is becoming more prevalent, the cost of image storage becomes factor to consider. The purpose of this study was to test the effect of reducing the tissue size of frequently scanned slides on digital whole slide image file size and file storage cost. We investigated the file sizes of our most commonly scanned slides – the daily special stain controls. The average file size of these stains ranges from 109 to 476 MB. All of the Masson trichrome slides, Movat pentachrome slides, and PAS slides were reviewed from 2015, and the tissue measurements and file sizes were recorded. New, smaller control tissue blocks were created. Once scanned, the tissue measurements and file sizes were also recorded. Overall, we reduced the size of the tissue on the slide by an average of 74%. We calculate that for these three control tissues alone, $3465 in storage costs could be saved every year by reducing the size of the tissue on the slide.

A Functional Variant of SMAD4 Enhances Thoracic Aortic Aneurysm and Dissection Risk through Promoting Smooth Muscle Cell Apoptosis and Proteoglycan Degradation

Recent studies indicate important roles for SMAD4 in SMCs proliferation, extracellular matrix maintenance, and blood vessel remodeling. However, the genetic effects of SMAD4 in the pathogenesis of thoracic aortic aneurysm and dissection (TAAD) are still largely unknown. Here we identified a functional variant of SMAD4 which might be involved in the pathological progression of TAAD. Five tagging SNPs of SMAD4 were genotyped in 202 TAAD cases and 400 controls using MALDI-TOF. rs12455792 CT or TT variant genotypes was associated with an significantly elevated TAAD risk (adjusted OR=1.58, 95%CI=1.09–2.30) under a dominant genetic model. It was located in the 5’UTR and predicted to influence transcription activity and RNA folding of SMAD4. In luciferase reporter assay, rs12455792 T allele markedly decreased luciferase activities. Accordingly, SMAD4 expression in tissues was lower in patients with CT or TT genotypes, compared with CC. Movat's pentachrome showed that rs12455792 T allele enhanced SMCs loss and fibers accumulation. With angiotensin II induction, rate of Apoptotic SMCs was significantly higher while SMAD4 silenced. Moreover, rs12455792 T allele also increased Versican degradation via ADAMTS-4. In conclusion, this variant might promote SMCs apoptosis and proteoglycans degradation, and further facilitate the progress of TAAD. Our findings identified rs12455792 as a predictor for progression of vascular media pathological changes related thoracic aortic disorders.

Application of omentum as an in vivo bioreactor for regeneration of decellularized human internal mammary artery.

This study investigates the extent of cell seeding as well as lumen patency in acellular Internal mammary artery (IMA) scaffolds that have been re-cellularized by omentum as a natural bioreactor. Sixteen Virgin female Wistar rats were selected for implantation of acellular scaffold in omentum. Following laparotomy omentum was retracted to the outside of the abdomen and the more vascularized portion of it was selected for the experiment. The scaffold was wrapped by omentum and placed between two layers of rectus muscles that have been previously dissected. Samples were taken from scaffolds at 2 and 3 months for histopathological evaluation. All the grafts were explanted at 2 and 3 months and the lumens were completely patent compared to the native scaffold. The histology of implanted IMA after 2 and 3 months showed progressive recellularization especially by smooth muscle cells over the media layer. CD 34 staining was positive adjacent to the grafts which showed well angiogenesis. Trichrome and Movat's Pentachrome staining showed normal collagen formation in the inner media layer. Promising results obtained from the introduced protocol for in vivo recellularization, including patent lumen and proper cell seeding, encourages application of the mentioned technique for experimental vascular graft application. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2685-2693, 2017.

Decellularization of Human Internal Mammary Artery: Biomechanical Properties and Histopathological Evaluation.

This study undertook to create small-diameter vascular grafts and assess their structure and mechanical properties to withstand arterial implantation. Twenty samples of intact human internal mammary arteries (IMAs) were collected and decellularized using detergent-based methods. To evaluate residual cellular and extracellular matrix (ECM) components, histological analysis was performed. Moreover, collagen typing and ECM structure were analyzed by Picrosirius red and Movat's pentachrome staining. Scanning electron microscopy was also applied to assess microarchitecture of both endothelial and adventitial surfaces of native and decellularized arterial samples. Furthermore, mechanical tests were performed to evaluate the rigidity and suture strength of the arteries. Human IMAs were completely decellularized in all three segments (proximal, middle, and distal). ECM proteins such as collagen and elastic fibers were efficiently preserved and no structural distortion in intima, media, and adventitial surfaces was observed. The parameters of the mechanical tests revealed no significant differences in the mechanical properties of decellularized arteries in comparison to native arteries with considerable strength, suture retention, and stress relaxation (Young's modulus [MPa] = 0.22 ± 0.023 [native] and 0.22 ± 0.015 [acellular]; and suture strength 0.56 ± 0.19 [native] vs. 0.56 ± 0.12 [acellular], respectively). Decellularized IMA represents a potential arterial scaffold as an alternative to autologous grafts for future arterial bypass surgeries. By this technique, microarchitecture and mechanical integrity of decellularized arteries were considerably similar to native arteries. The goal of this study was to introduce an efficient method for complete decellularization of human IMA and evaluate the ECM and biomechanical properties.

Assessment of atherosclerotic luminal narrowing of coronary arteries based on morphometrically generated visual guides.

Determination of the degree of stenosis of atherosclerotic coronary arteries is an important part of postmortem examination of the heart, but, unfortunately, estimation of the degree of luminal narrowing can be imprecise and tends to be approximations. Visual guides can be useful to assess this, but earlier attempts to develop such guides did not employ digital technology. Using this approach, we have developed two computer-generated morphometric guides to estimate the degree of luminal narrowing of atherosclerotic coronary arteries. The first is based on symmetric or eccentric circular or crescentic narrowing of the vessel lumen and the second on either slit-like or irregularly shaped narrowing of the vessel lumens. Using the Aperio ScanScope XT at a magnification of 20× we created digital whole-slide images of 20 representative microscopic cross sections of the left anterior descending (LAD) coronary artery, stained with either hematoxylin and eosin (H&E) or Movat's pentachrome stain. These cross sections illustrated a variety of luminal profiles and degrees of stenosis. Three representative types of images were selected and a visual guide was constructed with Adobe Photoshop CS5. Using the "Scale" and "Measurement" tools, we created a series of representations of stenosis with luminal cross sections depicting 20%, 40%, 60%, 70%, 80%, and 90% occlusion of the LAD branch. Four pathologists independently reviewed and scored the degree of atherosclerotic luminal narrowing based on our visual guides. In addition, digital technology was employed to determine the degree of narrowing by measuring the cross-sectional area of the 20 microscopic sections of the vessels, first assuming no narrowing and then comparing this to the percent of narrowing determined by precise measurement. Two of the observers were very experienced general autopsy pathologists, one was a first-year pathology resident on his first rotation on the autopsy service, and the fourth observer was a highly experienced cardiovascular pathologist. Interobserver reliability was assessed by determination of the intraclass correlation coefficient. The degrees of agreement for two H&E- and Movat-stained sections of the LADs from each of 10 decedents were 0.874 and 0.899, respectively, indicating strong interobserver agreement. On the average, the mean visual scores were ~8% less than the morphometric assessment (52.7 vs. 60.2), respectively. The visual guides that we have generated for scoring atherosclerotic luminal narrowing of coronary arteries should be helpful for a broad group of pathologists, from beginning pathology residents to experienced cardiovascular pathologists.

Stenting of Spontaneous Coronary Artery Dissection From a Pathological Point of View.

A 32-year-old female recently postpartum was admitted with chest pain and shortness of breath. She was diagnosed with acute inferior myocardial infarction and taken to catheterization laboratory in cardiogenic shock. The proximal right coronary artery was occluded, and an everolimus-eluting stent (Abbott Vascular, Santa Clara, CA) was implanted successfully. Left coronary angiography showed 50% stenosis of proximal left anterior descending (LAD) and total occlusion of left circumflex. The patient remained in cardiogenic shock despite insertion of an intra-aortic balloon pump and temporary pacemaker and died after cardiac arrest. At autopsy (Figure [A]), coronary arteries showed no gross atherosclerosis, but diffuse coronary artery dissections, involving left main, LAD, left circumflex, and right coronary artery. The stent was implanted in the true lumen (TL, Figure [A3 through A5]), though it was not well expanded and the underlying dissection was still observed. The dissection in the right coronary artery extended both proximally and distally, but severe narrowing was only observed in the distal right …

Loss of mechanical directional dependency of the ascending aorta with severe medial degeneration.

Biomechanical characterization of the aortic wall may help risk stratify patients with aneurysms. We investigated the degree of anisotropy, the directional dependency of mechanical properties, in control and aneurysmal ascending aortic tissue. We hypothesized that medial degeneration and aortic wall remodeling as found in aneurysmal tissue alter energy loss in both the circumferential and longitudinal directions, thereby reducing anisotropy. Aneurysmal and control ascending aortic tissue excised during surgery was subjected to biaxial tensile testing. Stress-strain relationships were collected in the circumferential and longitudinal directions; from these data, the mechanical properties of energy loss and the apparent modulus of elasticity were derived, and the associated anisotropy indices were calculated. Movat pentachrome histological staining was performed, and aortic wall medial degeneration was quantified. Energy loss was greater in the circumferential than the longitudinal direction, demonstrating significant anisotropy in both normal and aneurysmal aortas (P<.0001). This directional dependency diminished in (a) larger aortas (r2=0.15, P=.01), especially when indexed to body surface area (r2=0.29, P=.002); (b) aortas with greater overall energy loss (r2=0.44, P<.0001); (3) aortas associated with tricuspid valves (P=.004); and (4) higher collagen-to-elastin ratio (r2=0.29, P=.001). Aortas with collagen-to-elastin ratios greater than 2 were uniformly isotropic. Furthermore, the greatest energy loss anisotropy was found on the inner curvature of the aorta (P=.01). Energy loss demonstrates the directional dependency of aortic tissue. Energy loss isotropy is associated with medial degeneration, indicating that microstructural changes can be captured by global biomechanics, thereby identifying it as a marker of disease severity.

The investigation of BMP-2 and VEGF dual gene activated nanobone enhanced the vertebral bone of ovariec-tomized goat

Objective To verify BMP-2 and VEGF gene activated nanobone paste can effectively enhance the vertebral bone of ovariectomized goat. Methods From January, 2011 to May, 2016, the goats had been neutered by ovariectomy 6 months earlier to induceosteoporosis. Then surgery to established the model of vertebral bone defected with nanobone implanting, and the operation vertebrae divided randomly into 3 groups: control group, nanobone group and double gene activated nanobone group. Three months after operation the goats were sacrificed and removed the vertebrae. Micro CT analysis of micro three-dimensional structure of trabecular bone, scanning electron microscope (SEM) analysis of the two-dimensional structure of the vertebrae, the structure of trabecular bone was evaluated by movat pentachrome staining. Results The dual gene activated nanobone group compared with the nanobone group, the bone volume fraction (BV/TV) significantly increased (85% at 1.2 mm vs 43% at 1.2 mm, P < 0.05); the dual gene activated nanobone group compared with nanobone group, in the largest ROI (1.2 mm), TbTh increased 10.9% (374 ± 26.2 μm vs 337 ± 22.3 μm,P < 0.05); Trabecular distribution coefficient (TbPf) was significantly decreased (7.519 ± 0.184 mm-1 vs 7.529 ± 0.261 mm-1, P < 0.05); In the largest ROI (0.8 mm), trabecular distribution coefficient (TbPf) was significantly decreased (283 ± 36.4 μm vs 327 ± 31.2 μm, P < 0.05), In the largest ROI (0.8 mm), the trabecular bone volume (Tbn) was increased 20%(1.404 ± 0.283 mm-1vs 1.173 ± 0.224 mm-1, P < 0.05); Cortical thickness over the implantation area showed asignificant increase of 43% in vertebrae(P < 0.05); The histological analysis revealed a more extensive osseointegration of the dual gene activated nanobone group, with the presence of anabundant osteoid tissue and an osteoblastic celllining. Conclusion BMP-2 and VEGF gene activated nano bone paste can effectively enhance the vertebral bone of ovariectomized goat. Key words: BMP-2; VEGF; Nanobone; Ovariectomized; Goat; Vertebrae