Impact of prolonged storage time on homograft ultrastructures: an attempt to find optimal guidelines for homograft processing.

According to guidelines, total ischemic time for homografts at processing must be kept short to avoid degeneration. Many homografts are discarded due to practical inability to finish all steps from procurement to cryopreservation within the time limit. Although, several studies have shown that homografts with prolonged ischemic time show adequate quality and performance. Twenty aortic and 12 pulmonary homografts were collected and biopsies were retrieved at preparation (day 0) and after 1, 2, 3, 4, 7, 14, 21, 28, and 60 days in antibiotic decontamination at 4 °C. Biopsies were prepared for light microscopy (LM) and transmission electron microscopy (TEM). Assessment generated scores for cells, elastin, and collagen. Relative differences between times were compared with Wilcoxon signed rank test. Bonferroni corrected p value of 0.0056 was considered significant. LM could only reveal decrease in cell count at 60 days in aortic homografts, no other differences was detected. TEM showed affected cell appearance in day 3 and day 4 and beyond for aortic and pulmonary homografts respectively. Elastin appearance was affected at day 60 for aortic and day 21 for pulmonary homografts. Collagen appearance was affected at day 28 for aortic homografts, with no significant differences in pulmonary homografts. Cell degeneration starts early after homograft procurement, but elastic and collagen fibers are more resistant to degeneration. Overall structure integrity as seen in LM was not affected at all, while TEM could reveal small degeneration signs in individual elastic fibers and collagen bundles at 21 and 28 days respectively.

Ciprofloxacin accelerates aortic enlargement and promotes dissection and rupture in Marfan mice.

OBJECTIVE: Aortic aneurysm and dissection are major life-threatening complications of Marfan syndrome. Avoiding factors that promote aortic damage is critical in managing the care of these patients. Findings from clinical and animal studies raise concerns regarding fluoroquinolone use in patients at risk for aortic aneurysm and dissection. Therefore, we examined the effects of ciprofloxacin on aortic aneurysm and dissection development in Marfan mice. METHODS: Eight-week-old Marfan mice (Fbn1C1041G/+) were given ciprofloxacin (100 mg/kg/d; n = 51) or vehicle (n = 59) for 4 weeks. Mice were monitored for 16 weeks. Aortic diameters were measured by using ultrasonography, and aortic structure was examined by using histopathologic and immunostaining analyses. RESULTS: Vehicle-treated Fbn1C1041G/+ mice showed progressive aortic enlargement, with aortic rupture occurring in 5% of these mice. Compared with vehicle-treated Fbn1C1041G/+ mice, ciprofloxacin-treated Fbn1C1041G/+ mice showed accelerated aortic enlargement (P = .01) and increased incidences of aortic dissection (25% vs 47%, P = .03) and rupture (5% vs 25%, P = .005). Furthermore, ciprofloxacin-treated Fbn1C1041G/+ mice had higher levels of elastic fiber fragmentation, matrix metalloproteinase expression, and apoptosis than did vehicle-treated Fbn1C1041G/+ mice. CONCLUSIONS: Ciprofloxacin accelerates aortic root enlargement and increases the incidence of aortic dissection and rupture in Marfan mice, partially by suppressing lysyl oxidase expression and further compromising the inherited defect in aortic elastic fibers. Our findings substantiate that ciprofloxacin should be avoided in patients with Marfan syndrome.

Mutations in EPHB4 cause human venous valve aplasia.

Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex "organization" of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound, we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter in mice to study expression of its ligand, ephrinB2, and analyzed developmental phenotypes after conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.

Effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior.

Due to an increasing number of cardiovascular diseases, artificial heart valves and blood vessels have been developed. Although cardiovascular applications using decellularized tissue have been studied, the mechanisms of their functionality remain unknown. To determine the important factors for preparing decellularized cardiovascular prostheses that show good in vivo performance, the effects of the luminal surface structure of the decellularized aorta on thrombus formation and cell behavior were investigated. Various luminal surface structures of a decellularized aorta were prepared by heating, drying, and peeling. The luminal surface structure and collagen denaturation were evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining, and scanning electron microscopy (SEM) analysis. To evaluate the effects of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting tests and recellularization of endothelial cells and smooth muscle cells were performed. The results of the blood clotting test showed that the closer the luminal surface structure is to the native aorta, the higher the anti-coagulant property. The results of the cell seeding test suggest that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation, and functional expression accordingly. These results provide important factors for preparing decellularized cardiovascular prostheses and will lead to future developments in decellularized cardiovascular applications.

Adverse cardiovascular effects of exposure to cadmium and mercury alone and in combination on the cardiac tissue and aorta of Sprague-Dawley rats.

The aim of this study was to identify cardiovascular effects of relevant concentrations of Cd and Hg alone and in combination as a mixture in water. This was achieved by administering to male Sprague-Dawley rats via gavage 0.62 mg/kg Cd or 1.23 mg/kg Hg, or a combination of 0.62 mg/kg Cd and 1.23 mg/kg Hg in the co-exposure group for 28 days. Concentrations were the rat equivalence dosages of 1,000 times the World Health Organization's limits of 0.003 mg/L and 0.006 mg/L for Cd and Hg, respectively, for water. With termination, blood levels of the metals were increased. For all metal exposed groups, histological evaluation and transmission electron microscopy of the myocardium revealed myofibrillar necrosis, increased fibrosis, vacuole formation and mitochondrial damage. Cd caused the most mitochondrial damage while Hg to a greater degree induced fibrosis. In the aorta, both Cd and Hg also increased collagen deposition adversely altering the morphology of the fenestrated elastic fibers in the tunica media. Co-exposure resulted in increased cardiotoxicity with increased mitochondrial damage, fibrosis and distortion of the aortic wall as a result of increased collagen deposition, as well as altered elastin deposition, fragmentation and interlink formation. These are typical features of oxidative damage that correlates with a phenotype of premature ageing of the CVS that potentially can lead to hypertension and premature cardiac failure.

Evolving structure-function relations during aortic maturation and aging revealed by multiphoton microscopy.

The evolving microstructure and mechanical properties that promote homeostasis in the aorta are fundamental to age-specific adaptations and disease progression. We combine ex vivo multiphoton microscopy and biaxial biomechanical phenotyping to quantify and correlate layer-specific microstructural parameters, for the primary extracellular matrix components (fibrillar collagen and elastic lamellae) and cells (endothelial, smooth muscle, and adventitial), with mechanical properties of the mouse aorta from weaning through natural aging up to one year. The aging endothelium was characterized by progressive reductions in cell density and altered cellular orientation. The media similarly showed a progressive decrease in smooth muscle cell density and alignment though with inter-lamellar widening from intermediate to older ages, suggesting cell hypertrophy, matrix accumulation, or both. Despite not changing in tissue thickness, the aging adventitia exhibited a marked thickening and straightening of collagen fiber bundles and reduction in cell density, suggestive of age-related remodeling not growth. Multiple microstructural changes correlated with age-related increases in circumferential and axial material stiffness, among other mechanical metrics. Because of the importance of aging as a risk factor for cardiovascular diseases, understanding the normal progression of structural and functional changes is essential when evaluating superimposed disease-related changes as a function of the age of onset.

Increased Risk of Aortic Dissection with Perlecan Deficiency.

Perlecan (HSPG2), a basement membrane-type heparan sulfate proteoglycan, has been implicated in the development of aortic tissue. However, its role in the development and maintenance of the aortic wall remains unknown. Perlecan-deficient mice (Hspg2-/--Tg: Perl KO) have been found to show a high frequency (15-35%) of aortic dissection (AD). Herein, an analysis of the aortic wall of Perl KO mice revealed that perlecan deficiency caused thinner and partially torn elastic lamina. Compared to the control aortic tissue, perlecan-deficient aortic tissue showed a significant decrease in desmosine content and an increase in soluble tropoelastin levels, implying the presence of immature elastic fibers in Perl KO mice. Furthermore, the reduced expression of the smooth muscle cell contractile proteins actin and myosin in perlecan-deficient aortic tissue may explain the risk of AD. This study showed that a deficiency in perlecan, which is localized along the elastic lamina and at the interface between elastin and fibrillin-1, increased the risk of AD, largely due to the immaturity of extracellular matrix in the aortic tissue. Overall, we proposed a new model of AD that considers the deficiency of extracellular molecule perlecan as a risk factor.

The microscopic anatomy of endothelial cells in human atherosclerosis: Focus on ER and mitochondria.

Once regarded merely as a bland lipid storage disease consequence of aging, atherosclerosis is currently considered a slow and continuous inflammatory process (partially controllable by treatment) with complex etiology involving a multitude of genetic and environmental risk factors which ultimately result in the formation of the plaque. The vascular endothelium, a monolayer of endothelial cells (ECs), is an important regulatory "organ" critical for cardiovascular homeostasis in health which also contributes significantly to the pathomechanisms of several disease states, including atherosclerosis. Over the years, there has been evidence highlighting the central role of endoplasmic reticulum (ER) in the maintenance of endothelial function and perturbations in ER biology have been proposed to adversely affect a diverse range of endothelial functions. Of particular interest is the evidence that under certain pathophysiological circumstances, abnormal ER ultrastructure correlates with altered ER function and signaling and can contribute to cell injury and apoptosis. Therefore, the ultrastructural traits of ER membranes can have important implications not only for their functional bearings but also for the etiology and pathophysiology of diverse human disorders. With regard to atherosclerosis, the focus of ER research has been centered on the molecular signals originated from the ER to manage conditions of stress, leaving the fine structure of this organelle an almost unexplored (but promising) area of studies. There is, also, increasing evidence that mitochondrial dysfunction plays a critical role in promoting cell apoptosis, inflammation, and oxidative stress, thereby contributing to atheroma growth. It is within this context that the present study has been undertaken to investigate the microscopic architecture of ECs in human atherosclerosis and to determine whether the potential structural abnormalities of ER and mitochondria may play a central pathogenic role in atherogenesis or may merely reflect the condition of a tissue whose integrity has already been disturbed or destroyed. For this purpose, transmission electron microscopy (TEM) remains a powerful technique that can not only provide information about the ultrastructural state of cell organelles but also allow the correlation between different subcellular alterations indicative of a certain pathophysiological condition and cellular response. The present study expands the spectrum of ultrastructural defects known to exist in human atherosclerosis and suggests that ER alterations may be of great importance in the pathogenesis of the disease. The architectural changes of ER may be considered early pathological events that precede any overt histologic abnormalities in the vascular endothelium and its subcellular organelles, primarily the mitochondrial pool.

Suppression of type 2 diabetes mellitus-induced aortic ultrastructural alterations in rats by insulin: an association of vascular injury biomarkers.

Diabetes represents a major public health problem and an estimated 70% of people with diabetes die of cardiovascular complications. The protective effect of insulin treatment against ultrastructural damage to the tunica intima and tunica media of the aorta induced by type 2 diabetes mellitus (T2DM) has not been investigated before using transmission electron microscopy (TEM). Therefore, we induced T2DM in rats using high fat diet and streptozotocin (50 mg/kg) and administered insulin daily by i.v injection for 8 weeks to the treatment group. Whereas, the T2DM control group were left untreated for the duration of the experiment. A comparison was also made between the effect of insulin on aortic tissue and the blood level of biomarkers of vascular injury, inflammation, and oxidative stress. T2DM induced profound ultrastructural damage to the aortic endothelium and vascular smooth muscle cells, which were substantially protected with insulin. Furthermore, insulin returned blood sugar to a control level and significantly (p < .05) inhibited diabetic up-regulation of endothelial and leukocyte intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), endothelial cell adhesion molecules, P-selectin and E-selectin, tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), and malondialdehyde (MDA). Furthermore, insulin augmented the blood level of the anti-oxidant enzyme superoxide dismutase (SOD). We conclude that in a rat model of T2DM, insulin treatment substantially reduces aortic injury secondary to T2DM for a period of 8 weeks, possibly due to the inhibition of hyperglycemia, vascular activation, inflammation, and oxidative stress.

Cav-1 (Caveolin-1) Deficiency Increases Autophagy in the Endothelium and Attenuates Vascular Inflammation and Atherosclerosis.

OBJECTIVE: Endothelial Cav-1 (caveolin-1) expression plays a relevant role during atherogenesis by controlling NO production, vascular inflammation, LDL (low-density lipoprotein) transcytosis, and extracellular matrix remodeling. Additional studies have identified cholesterol-rich membrane domains as important regulators of autophagy by recruiting ATGs (autophagy-related proteins) to the plasma membrane. Here, we investigate how the expression of Cav-1 in the aortic endothelium influences autophagy and whether enhanced autophagy contributes to the atheroprotective phenotype observed in Cav-1-deficient mice. Approach and Results: To analyze the impact of Cav-1 deficiency on regulation of autophagy in the aortic endothelium during the progression of atherosclerosis, we fed Ldlr-/- and Cav-1-/-Ldlr-/- mice a Western diet and assessed autophagy in the vasculature. We observe that the absence of Cav-1 promotes autophagy activation in athero-prone areas of the aortic endothelium by enhancing autophagic flux. Mechanistically, we found that Cav-1 interacts with the ATG5-ATG12 complex and influences the cellular localization of autophagosome components in lipid rafts, which controls the autophagosome formation and autophagic flux. Pharmacological inhibition of autophagy attenuates the atheroprotection observed in Cav-1-/- mice by increasing endothelial inflammation and macrophage recruitment, identifying a novel molecular mechanism by which Cav-1 deficiency protects against the progression of atherosclerosis. CONCLUSIONS: These results identify Cav-1 as a relevant regulator of autophagy in the aortic endothelium and demonstrate that pharmacological suppression of autophagic flux in Cav-1-deficient mice attenuates the atheroprotection observed in Cav-1-/- mice. Additionally, these findings suggest that activation of endothelial autophagy by blocking Cav-1 might provide a potential therapeutic strategy for cardiovascular diseases including atherosclerosis.

Vanadium inhibits Type 2 diabetes mellitus-induced aortic ultrastructural alterations associated with the Inhibition of dyslipidemia and biomarkers of inflammation in rats / El vanadio inhibe las alteraciones ultraestructurales aórticas inducidas por la diabetes mellitus Tipo 2 asociadas con la inhibición de la dislipidemia y los biomarcadores de inflamación en ratas

The potential inhibitory effect of the insulin mimicking agent, vanadium on type 2 diabetes mellitus (T2DM)induced alterations to the aorta ultrastructure associated with the suppression of dyslipedima and biomarkers of inflammation has not been investigated before. Therefore, we tested whether vanadium can protect against aortic injury induced secondary to T2DM possibly via the inhibition of blood lipid and inflammatory biomarkers. T2DM was induced in rats by a high-fat diet and streptozotocin (50 mg/ kg), and the treatment group started vanadium treatment five days post diabetic induction and continued until being sacrificed at week 10. Using light and electron microscopy examinations, we observed in the model group substantial damage to the aorta tissue such as damaged endothelium, degenerative cellular changes with vacuolated cytoplasm and thickened internal elastic lamina that were substantially ameliorated by vanadium. Administration of vanadium to diabetic rats also significantly (p<0.05) reduced blood levels of glucose, hyperlipidemia and biomarkers of inflammation (TNF-a, IL-6). We conclude that vanadium protects against T2DM-induced aortic ultrastructural damage in rats, which is associated with the inhibition of blood sugar and lipid and inflammatory biomarkers.

El potencial efecto inhibidor del agente imitador de la insulina, el vanadio en las alteraciones inducidas por la diabetes mellitus tipo 2 (DM2) en la ultraestructura de la aorta, asociada con la supresión de dislipidemia y los biomarcadores de inflamación no se ha investigado anteriormente. El objetivo fue estudiar las propiedades del vanadio para proteger contra la lesión aórtica inducida a la DM2, a través de la inhibición de los lípidos sanguíneos y los biomarcadores inflamatorios. La DM2 fue inducida en ratas con una dieta alta en grasas y estreptozotocina (50 mg / kg), y el grupo de tratamiento fue sometido a un régimen continuo con vanadio, cinco días después de la inducción diabética hasta ser sacrificadas en la semana 10. Se utilizaron exámenes de luz y microscopía electrónica en el grupo modelo y se observó un daño sustancial al tejido de la aorta, como también en el endotelio; los cambios celulares degenerativos con citoplasma vacuolado y lámina elástica interna engrosada mejoró sustancialmente con vanadio. La administración de vanadio a ratas diabéticas también redujo significativamente (p <0,05) los niveles sanguíneos de la glucosa, hiperlipidemia y los biomarcadores de inflamación (TNFa, IL-6). En conclusión, el vanadio protege contra el daño ultraestructural aórtico inducido por T2DM en ratas, que es asociado con la inhibición del azúcar en la sangre y los biomarcadores de lípidos y de inflamatorios.

Connective Tissue Ultrastructure: A Direct Comparison between Conventional Specimen Preparation and High-Pressure Freezing/Freeze-Substitution.

It is generally agreed within the microscopy community that the quality of ultrastructure within the connective tissue matrix resulting from high-pressure freezing followed by freeze-substitution (HPF/FS) far exceeds that gained following the "conventional" preparation method, which includes aqueous fixation, dehydration, and embedding. Exposure to cryogen at high pressure is the only cryopreservation method capable of vitrifying tissue structure to a depth exceeding 200 µm. Cells within connective tissues prepared by HPF/FS are universally larger, filling the commonly seen void at the juncture between cell and matrix. Without significant shrinkage of cells and the coincident extraction of the cytosolic components, well-resolved organelles are less clustered within an expanded cytosol. Much of the artifact from "conventional" methods occurs as large space filling and also smaller fibril-associated proteoglycans are extracted during fixation. However, the visualization of some matrix features by electron microscopy is actually dependent on the collapse or extraction of these "masking" components. Herein, we argue that an impression of ultrastructure within commonly studied matrices, in particular skin, is best gained following the evaluation of both conventional preparations and tissue prepared by HPF/FS. Anat Rec, 2019. © 2019 American Association for Anatomy.

Sildenafil reduces aortic endothelial dysfunction and structural damage in spontaneously hypertensive rats: Role of NO, NADPH and COX-1 pathways.

Arterial hypertension is a condition associated with endothelial dysfunction, accompanied by an imbalance in the production of reactive oxygen species (ROS) and NO. The aim of this study was to investigate and elucidate the possible mechanisms of sildenafil, a selective phosphodiesterase-5 inhibitor, actions on endothelial function in aortas from spontaneously hypertensive rats (SHR). SHR treated with sildenafil (40 mg/kg/day, p.o., 3 weeks) were compared to untreated SHR and Wistar-Kyoto (WKY) rats. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and vascular reactivity was determined in isolated rat aortic rings. Circulating endothelial progenitor cells and systemic ROS were measured by flow cytometry. Plasmatic total antioxidant capacity, NO production and aorta lipid peroxidation were determined by spectrophotometry. Scanning electron microscopy was used for structural analysis of the endothelial surface. Sildenafil reduced high SBP and partially restored the vasodilator response to acetylcholine and sodium nitroprusside in SHR aortic rings. Using selective inhibitors, our experiments revealed an augmented participation of NO, with a simultaneous decrease of oxidative stress and of cyclooxygenase-1 (COX-1)-derived prostanoids contribution in the endothelium-dependent vasodilation in sildenafil-treated SHR compared to non-treated SHR. Also, the relaxant responses to sildenafil and 8-Br-cGMP were normalized in sildenafil-treated SHR and sildenafil restored the pro-oxidant/antioxidant balance and the endothelial architecture. In conclusion, sildenafil reverses endothelial dysfunction in SHR by improving vascular relaxation to acetylcholine with increased NO bioavailability, reducing the oxidative stress and COX-1 prostanoids, and improving cGMP/PKG signaling. Also, sildenafil reduces structural endothelial damage. Thus, sildenafil is a promising novel pharmacologic strategy to treat endothelial dysfunction in hypertensive states reinforcing its potential role as adjuvant in the pharmacotherapy of cardiovascular diseases.

Metformin suppresses aortic ultrastrucural damage and hypertension induced by diabetes: a potential role of advanced glycation end products.

Cardiovascular disease secondary to diabetes represents a significant challenge to the health community. The advanced glycation end products (AGEs) play an important role in diabetes-mediated vascular injury. We tested whether metformin can suppress aortic AGEs production and protect against aortic injuries (aortopathy) and hypertension in streptozotocin-induced type 2 diabetes mellitus (T2DM) animal model. T2DM was induced in rats two weeks after being fed on a high carbohydrate and fat diet (HCFD), and continued on a HCFD until being sacrificed at week 12 (model group). The protective group was put on metformin two weeks before diabetic induction and continued on metformin and HCFD until the end of the experiment, at week 12. Using electron microscopy examinations, we observed in the model group substantial damage to the ultrastructure of aortic endothelial and vascular smooth muscle layers as demonstrated by markedly distorted vacuolated endothelial and vascular smooth muscle cells with pyknotic nuclei detached from the underlying basement membrane, which were protected by metformin. Also, metformin significantly (p < .05) decreased both systolic and diastolic blood pressure, aortic levels of AGEs, and blood levels of oxidative stress and inflammatory biomarkers. We conclude that metformin protects against T2DM-induced aortopathy and hypertension, possibly via the inhibition of AGEs, inflammation, and oxidative stress.

c-Kit suppresses atherosclerosis in hyperlipidemic mice.

Atherosclerosis is the most common underlying cause of cardiovascular morbidity and mortality worldwide. c-Kit (CD117) is a member of the receptor tyrosine kinase family, which regulates differentiation, proliferation, and survival of multiple cell types. Recent studies have shown that c-Kit and its ligand stem cell factor (SCF) are present in arterial endothelial cells and smooth muscle cells (SMCs). The role of c-Kit in cardiovascular disease remains unclear. The aim of the current study is to determine the role of c-Kit in atherogenesis. For this purpose, atherosclerotic plaques were quantified in c-Kit-deficient mice (KitMut) after they were fed a high-fat diet (HFD) for 16 wk. KitMut mice demonstrated substantially greater atherosclerosis compared with control (KitWT) littermates (P < 0.01). Transplantation of c-Kit-positive bone marrow cells into KitMut mice failed to rescue the atherogenic phenotype, an indication that increased atherosclerosis was associated with reduced arterial c-Kit. To investigate the mechanism, SMC organization and morphology were analyzed in the aorta by histopathology and electron microscopy. SMCs were more abundant, disorganized, and vacuolated in aortas of c-Kit mutant mice compared with controls (P < 0.05). Markers of the "contractile" SMC phenotype (calponin, SM22α) were downregulated with pharmacological and genetic c-Kit inhibition (P < 0.05). The absence of c-Kit increased lipid accumulation and significantly reduced the expression of the ATP-binding cassette transporter G1 (ABCG1) necessary for lipid efflux in SMCs. Reconstitution of c-Kit in cultured KitMut SMCs resulted in increased spindle-shaped morphology, reduced proliferation, and elevated levels of contractile markers, all indicators of their restored contractile phenotype (P < 0.05).NEW & NOTEWORTHY This study describes the novel vasculoprotective role of c-Kit against atherosclerosis and its function in the preservation of the SMC contractile phenotype.

An innovative method to obtain porous porcine aorta scaffolds for tissue engineering.

Decellularized porcine aorta (PA) is a promising biomaterial for vascular substitutes. However, decellularized PAs suffer from mechanical weakness and have less pores, which limit cellular ingrowth into the grafts and hinder the remodeling. In this study, PAs were decellularized by vacuum-freeze-thawing cycles and 0.3% of sodium dodecyl sulfate (SDS) buffer (VLS). Results showed that the application of vacuum-freeze-thawing significantly improved the decellularization efficiency of SDS while effectively preserved the mechanical function of PA tissues, decreased residual SDS, and minimized cytotoxicity. Furthermore, scanning electron microscopy (SEM) examination demonstrated that VLS generated interconnected pores with uniform distribution. In vivo subcutaneous implantation assay further demonstrated that VLS implants had less calcification and adverse inflammatory response. Moreover, VLS treatment markedly enhanced ingrowth of myofibroblasts and endothelial cells, and thereby promoted synthesis of extracellular matrix and vascularization. These results suggest that the application of vacuum-freeze-thawing into the decellularization process may produce a promising vascular graft candidate for tissue engineering application.

Pre-diabetes induces ultrastructural alterations in the large blood vessel aorta in rats / La prediabetes induce alteraciones ultraestructurales en la aorta de los grandes vasos sanguíneos en ratas

Excessive consumption of carbohydrate and fat increases the risk of cardiovascular disease. We sought to determine the potential ultrastructural alterations in large blood vessels induced by a high fat and fructose diet (HFD) in a rat model of prediabetes. Rats were either fed with HFD (model group) or a standard laboratory chow (control group) for 15 weeks before being sacrificed. The harvested thoracic aorta tissues were examined using transmission electron microscopy (TEM), and blood samples were assayed for biomarkers of pre-diabetes.TEM images showed that HFD induced profound pathological changes to the aortic wall layers, tunica intima and tunica media ultrastructures in the pre-diabetic rats as shown by apoptotic endothelial cells with pyknotic nuclei, damaged basal lamina, deteriorated smooth muscle cells that have irregular plasma membranes, shrunken nucleus with clumped nuclear chromatin, damaged mitochondria and few cytoplasmic lipid droplets and vacuoles. In addition, HFD significantly (p<0.05) decreased adiponectin and increased biomarkers of lipidemia, glycaemia, inflammation, oxidative stress, vascular injury such as soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion protein 1 (sVCAM-1), endothelin-1 (ET-1), and coagulation and thrombosis such as Von Willebrand factor (vWF), and plasminogen activator inhibitor-1 (PAI-1), compared to normal levels of these parameters in the control group. Thus, we demonstrated that feeding rats with a HFDisable to develop a pre-diabetic animal model that is useful to study the aortic ultrastructural alterations.

El consumo excesivo de carbohidratos y grasas aumenta el riesgo de enfermedades cardiovasculares. Intentamos determinar las posibles alteraciones ultraestructurales en los grandes vasos sanguíneos, inducidas por una dieta alta en grasas y fructosa (HFD) en un modelo de rata de prediabetes. Las ratas se alimentaron con HFD (grupo modelo) o una comida de laboratorio estándar (grupo de control) durante 15 semanas antes de ser sacrificadas. Los tejidos de la aorta torácica recolectados se examinaron mediante microscopía electrónica de transmisión (TEM) y las muestras de sangre se analizaron para detectar biomarcadores de prediabetes. Las imágenes TEM mostraron que HFD indujo cambios patológicos profundos en las capas de la pared aórtica, túnica íntima y túnica media en la ratas pre-diabéticas como lo muestran las células endoteliales apoptóticas con núcleos picnóticos, lámina basal dañada, células musculares lisas deterioradas que tienen membranas plasmáticas irregulares, núcleo encogido con cromatina nuclear aglomerada, mitocondrias dañadas y pocas gotitas lipídicas citoplásmicas y vacuolas. Además, HFD presentó disminución significativa de adiponectina (p <0,05), y aumento de biomarcadores de lipidemia, glucemia, inflamación, estrés oxidativo, lesión vascular como la molécula de adhesión intercelular soluble 1 (sICAM-1), proteína de adhesión de células vasculares soluble 1 (sVCAM-1), endotelina 1 (ET-1), y la coagulación y la trombosis, como el factor de Von Willebrand (vWF), y el inhibidor del activador del plasminógeno-1 (PAI -1), en comparación con los niveles normales de estos parámetros en el grupo de control. Por tanto, la alimentación de ratas con HFD es capaz de desarrollar un modelo animal prediabético que es útil para estudiar las alteraciones ultraestructurales aórticas.

Improved matrix coating for positive- and negative-ion-mode MALDI-TOF imaging of lipids in blood vessel tissues.

High-quality matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) of lipids in biological tissue relies on the fabrication of a homogeneous matrix coating featuring best possible analyte integration. This communication addresses a matrix vapor deposition/recrystallization process for the application of 1,5-diaminonaphthalene (1,5-DAN) onto slices of human aortic tissue. The matrix coating is compatible with both positive- as well as negative-ion-mode MALDI MSI facilitating a significantly enhanced detection of lipid-related signals in different cell layers of blood vessel walls. Graphical abstract.

Some segmental morphological and morphometrical features of the intima and media of the aortic wall in Chinchilla lanigera.

BACKGROUND: The aim of this study is to describe the morphology, morphometry and ultrastructure of segments of the thoracic and abdominal aorta portions in Chinchilla lanigera. Thickness measurements of the tunica intima and media complex of the aorta were taken. MATERIALS AND METHODS: In all observed specimens, the thickness values for the tunica intima and media complex of the cranial thoracic aorta were significantly higher (mean: 702.19 µm) when compared to the values of other analysed aortic segments (means: 354.18 µm; 243.55 µm). Complex statistical methods were used to assess the differences between various aortic segments. RESULTS AND CONCLUSIONS: The components of the vessel walls show variations in structure and thickness, presumably due to an adaptation to functional demand.

Heart ventricular histology and microvasculature together with aortic histology and elastic lamellar structure: A comparison of a novel dual-purpose to a broiler chicken line.

The use of dual-purpose chickens is a strategy to avoid killing one-day-old male chicks of egg laying lines. Lohmann Dual (LD) is a novel dual-purpose chicken line created by the crossbreeding of layer and broiler lines. However, many of the cardiovascular diseases of broilers are likely to be associated with intensive genetic selection for growth and feed conversion efficiency. This study aimed to compare the macroscopic and microscopic structure of the heart and the aorta of the LD chicken line with that of the broiler chicken line, Ross 308 (Ross) under typical husbandry conditions for meat production. Eighty, one-day-old male chicks of each line were housed for 5 weeks (Ross) and 9 weeks (LD). Six birds of each line were sampled weekly. Heart mass, thickness of ventricular walls, cardiomyocyte size and blood capillary density as well as aortic diameter and thickness, number of elastic lamellae and elastic fiber percentage in the aortic wall were determined. The growth patterns of the heart were the same in the two lines. Although LD chickens had a lower absolute heart mass than that of Ross chickens, the relative heart mass in both lines was similar. The cardiomyocytes of LD chickens were larger than those of Ross's of the same body weight (BW), nevertheless both lines had similar thicknesses of their ventricular walls. The blood capillary density was greater in the LD heart than in that of the Ross heart. The aorta of LD chickens had proportionally; a greater aortic lumen radius, larger numbers of elastic lamellae and more elastic fibers than in Ross chickens. Our results suggest that the heart and aorta of the LD chickens have not been disadvantaged by their intensive genetic selection; furthermore, LD chickens have a better myocardial capillary supply and better aortic mechanical properties than those of Ross chickens.