Differences between inflammatory cells infiltrated into tunica intima, media, and adventitia of ascending aortic aneurysms within diabetic and hypertensive patients.

The risk factors that are the most significant for the development of most cardiovascular diseases are arterial hypertension (AH), type 2 diabetes (DM), and inflammation. However, for the development of aortic aneurysms, DM is not one of them. Our study aimed to evaluate the difference between inflammatory infiltration in three individual layers of the ascending aortic aneurysm within diabetic and hypertensive patients. Forty-five patients aged 36 to 80 were divided into a group with diabetic patients without AH (group DM, N=8) and hypertensive patients without DM (group AH, N=37). For the histological analysis, aortic aneurysms were stained with hematoxylin eosin and Movat. We used immunochemical methods to detect pro- (M1), anti-inflammatory (M2) macrophages, T-helper, T-killer cells, B cells, and plasma cells. Statistical analysis was done by independent-samples Kruskal-Wallis test adjusted by Bonferroni correction for multiple tests (P<0.05). We found no difference in the volume density of collagen, elastin, vascular smooth muscle cells (VSMC), and ground substance between groups. In the DM group, there were significantly fewer M2, T-helpers, and T-killers in the media than in the intima and the adventitia (P<0.05). There were no significant differences in the number of M1, B, and plasma cells between all three vascular layers (P<0.05). In the AH group, there were significantly fewer B and plasma cells, T-helper, T-killer cells, M1, and M2 in the media than in the intima and adventitia (P<0.05). Our results conclude that the tunica media in the aneurismal wall of the AH group retained immune privilege. In contrast, in the DM group, all three layers were immune-privileged.

Increased frequency of proangiogenic tunica intima endothelial kinase 2 (Tie2) expressing monocytes in individuals with type 2 diabetes mellitus.

BACKGROUND: Individuals with type 2 diabetes mellitus (T2DM) have an increased risk for developing macrovascular disease (MVD) manifested by atherosclerosis. Phenotypically and functionally different monocyte subsets (classical; CD14++CD16-, non-classical; CD14+CD16++, and intermediate; CD14++CD16+) including pro-angiogenic monocytes expressing Tie2 (TEMs) can be identified. Here we investigated monocyte heterogeneity and its association with T2DM and MVD. METHODS: Individuals with (N = 51) and without (N = 56) T2DM were recruited and allocated to "non-MVD" or "with MVD" (i.e., peripheral or coronary artery disease) subgroups. Blood monocyte subsets were quantified based on CD14, CD16 and Tie2 expression levels. Plasma levels of Tie2-ligands angiopoietin-1 and angiopoietin-2 were determined using ELISA. Carotid endarterectomy samples from individuals with (N = 24) and without (N = 22) T2DM were stained for intraplaque CD68+ macrophages (inflammation) and CD34+ (angiogenesis), as plaque vulnerability markers. RESULTS: Monocyte counts were similar between individuals with T2DM and healthy controls (non-diabetic, non-MVD). Non-classical monocytes were reduced (p < 0.05) in T2DM, whereas the percentage of TEMs within the intermediate subset was increased (p < 0.05). T2DM was associated with increased angiopoietin-1 (p < 0.05) and angiopoietin-2 (p = 0.0001) levels. Angiopoietin-2 levels were higher in T2DM individuals with MVD compared with non-MVD (p < 0.01). Endarterectomized plaques showed no differences in macrophage influx and microvessel number between individuals with and without T2DM. CONCLUSIONS: Monocyte subset distribution is altered in T2DM with reduced non-classical monocytes and increased TEM percentage in the intermediate monocyte subset. Increased angiopoietin-2 levels together with increased frequency of TEMs might promote plaque vulnerability in T2DM which could however not be confirmed at tissue level in advanced atherosclerotic lesions.

Impact of hepatitis virus infection on arterial calcification among incident hemodialysis patients / Impacto de la infección por el virus de la hepatitis en la calcificación arterial en nuevos pacientes en hemodiálisis

BACKGROUND: The impact of hepatitis virus infection on arterial calcification (AC) was not studied. OBJECTIVE: To study the prevalence, severity and distribution of AC in incident hemodialysis patients with hepatitis B and C viral infection. Cases and methods: 172 stage 5 CKD adults (98 male and 74 female) were included; 58 of them were seronegative for both hepatitis B and C (SN group), 48 were positive for hepatitis B virus infection (HBV group) and 66 were hepatitis C virus positive (HCV group). Beside histopathology of the obtained arterial samples, all these cases were examined for body mass index (BMI), serum calcium (Ca), phosphorus (P), alkaline phosphatase (AP), serum albumin, uric acid (UA), alanine transaminase (ALT), parathormone (PTH), fibroblast growth factor 23(FGF23), interleukin 6 (IL6), and 25 hydroxy vitamin D (25 (OH) vit D), hemoglobin concentration, and serum ferritin. RESULTS: 86 (50%) of the cases had AC; 11 of them were in SN group (19%), 9 in HBV group (18.8%) and all the 66 CV group (100%). In SN group, 4 had intimal calcification, 5 had medial calcification, and 2 had both intimal and medial calcification. In HBV group, 9 had intimal calcification, while no cases were encountered with either medial or both site calcifications. In HCV group, 16 had intimal calcification, 31 had medial calcification, and 19 had both intimal and medial calcification. Calcification was in the form of spots in one case in SN group, and 6 cases in HBV group, a single plaque of calcification in 5 cases of SN group, 3 cases of HBV group, and 16 cases of HCV group, multiple plaques were detected in 4 cases in SN group, and 31 cases in HCV group, and diffuse calcification in one case in SN group, and 19 cases in HCV group. In HBV group, calcification was only detected in patients with high viremia, while all patients with low or moderate viremia were devoid of calcification. In HCV group, all patients with low viremia had intimal solitary plaque of calcification, all patients with moderate viremia had multiple plaques of medial calcification, while all patients with high viremia had diffuse intimal and medial calcification. Both groups of viral hepatitis were significantly different in comparison to SN group in either distribution or calcification score (P < 0.001 in all). HBV group had significantly lower serum P, CaxP and PTH in comparison to SN group (4.6±0.66 vs. 5.45±0.77mg/dL, 36.4±7.2 vs. 44.1±8.69, and 348±65.4 vs. 405.9±83.2pg/mL, P<0.001, <0.001, and 0.035 respectively). On the other hand, HCV group did not show any significant difference in any of the studied parameters compared to SN group. CONCLUSION: HCV positive patients are more prone to develop AC that is more extensive. HBV positive patients were less likely to have arterial medial calcification, probably related to lower serum phosphorus, CaxP product and PTH. HCV infection should be added as risk factor for AC among CKD patients. Further studies are needed to confirm these findings

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Impact of hepatitis virus infection on arterial calcification among incident hemodialysis patients.

BACKGROUND: The impact of hepatitis virus infection on arterial calcification (AC) was not studied. OBJECTIVE: To study the prevalence, severity and distribution of AC in incident hemodialysis patients with hepatitis B and C viral infection. CASES AND METHODS: 172 stage 5 CKD adults (98 male and 74 female) were included; 58 of them were seronegative for both hepatitis B and C (SN group), 48 were positive for hepatitis B virus infection (HBV group) and 66 were hepatitis C virus positive (HCV group). Beside histopathology of the obtained arterial samples, all these cases were examined for body mass index (BMI), serum calcium (Ca), phosphorus (P), alkaline phosphatase (AP), serum albumin, uric acid (UA), alanine transaminase (ALT), parathormone (PTH), fibroblast growth factor 23(FGF23), interleukin 6 (IL6), and 25 hydroxy vitamin D (25 (OH) vit D), hemoglobin concentration, and serum ferritin. RESULTS: 86 (50%) of the cases had AC; 11 of them were in SN group (19%), 9 in HBV group (18.8%) and all the 66 HCV group (100%). In SN group, 4 had intimal calcification, 5 had medial calcification, and 2 had both intimal and medial calcification. In HBV group, 9 had intimal calcification, while no cases were encountered with either medial or both site calcifications. In HCV group, 16 had intimal calcification, 31 had medial calcification, and 19 had both intimal and medial calcification. Calcification was in the form of spots in one case in SN group, and 6 cases in HBV group, a single plaque of calcification in 5 cases of SN group, 3 cases of HBV group, and 16 cases of HCV group, multiple plaques were detected in 4 cases in SN group, and 31 cases in HCV group, and diffuse calcification in one case in SN group, and 19 cases in HCV group. In HBV group, calcification was only detected in patients with high viremia, while all patients with low or moderate viremia were devoid of calcification. In HCV group, all patients with low viremia had intimal solitary plaque of calcification, all patients with moderate viremia had multiple plaques of medial calcification, while all patients with high viremia had diffuse intimal and medial calcification. Both groups of viral hepatitis were significantly different in comparison to SN group in either distribution or calcification score (P<0.001 in all). HBV group had significantly lower serum P, CaxP and PTH in comparison to SN group (4.6±0.66 vs. 5.45±0.77mg/dL, 36.4±7.2 vs. 44.1±8.69, and 348±65.4 vs. 405.9±83.2pg/mL, P<0.001, <0.001, and 0.035 respectively). On the other hand, HCV group did not show any significant difference in any of the studied parameters compared to SN group. CONCLUSION: HCV positive patients are more prone to develop AC that is more extensive. HBV positive patients were less likely to have arterial medial calcification, probably related to lower serum phosphorus, CaxP product and PTH. HCV infection should be added as risk factor for AC among CKD patients. Further studies are needed to confirm these findings.

Microarray profiling analysis and validation of novel long noncoding RNAs and mRNAs as potential biomarkers and their functions in atherosclerosis.

Long noncoding (lnc)RNAs have been implicated in the development and progression of atherosclerosis. However, the expression and mechanism of action of lncRNAs in atherosclerosis are still unclear. We implemented microarray analysis in human advanced atherosclerotic plaques and normal arterial intimae to detect the lncRNA and mRNA expression profile. Gene Ontology functional enrichment and pathway analyses were applied to explore the potential functions and pathways involved in the pathogenesis of atherosclerosis. A total of 236 lncRNAs and 488 mRNAs were selected for further Ingenuity Pathway Analysis. Moreover, quantitative RT-PCR tests of most selected lncRNAs and mRNAs with high fold changes were consistent with the microarray data. We also performed ELISA to investigate the corresponding proteins levels of selected genes and showed that serum levels of SPP1, CD36, ATP6V0D2, CHI3L1, MYH11, and BDNF were differentially expressed in patients with coronary heart disease compared with healthy subjects. These proteins correlated with some biochemical parameters used in the diagnosis of cardiovascular diseases. Furthermore, receiver operating characteristic analysis showed a favorable diagnostic performance. The microarray profiling analysis and validation of differentially-expressed lncRNAs and mRNAs in atherosclerosis not only provide new insights into the pathogenesis of this disease but may also reveal new biomarkers for its diagnosis and treatment.

An autopsy case of pulmonary artery intimal sarcoma: detailed observation of tumor and its related lesions in pulmonary arteries.

We report an autopsy-proven case of a 33-year-old man who died of intimal sarcoma of the pulmonary artery. A large mass (5×4 cm) occluded the main and bilateral pulmonary arteries. Tumor cell morphology was consistent with that of undifferentiated pleomorphic sarcoma. Comprehensive histological observation of 18 pulmonary arteries from proximal to distal revealed continuous extension of the tumor from the main to the subsegmental arteries along the intima, forming an arteriosclerosis-like intimal thickening. Distal small arteries were also affected by eccentric intimal thickening or recanalization. Lung parenchyma was not involved, although there were two wedge-shaped small pulmonary infarctions caused by tumorous obstruction of the associated arteries. Histological results indicated that the intimal sarcoma in the pulmonary artery, which appeared occlusive with growth limited to the proximal artery, had in fact already spread more peripherally than expected. Both the proximal lesions and the distal small arteries were affected by peripheral tumor emboli or by pulmonary hypertension induced by the proximal tumor. However, as seen in this case, most of the occlusive tumor was located locally and intraluminally, in the proximal artery, and removing the proximal tumor by pulmonary endarterectomy was considered effective for symptomatic improvement.

Spectral fingerprinting of decellularized heart valve scaffolds.

Decellularized heart valves hold promise for their use as bioscaffolds in cardiovascular surgery. Quality assessment of heart valves after decellularization processing and/or storage is time consuming and destructive. Fourier transform infrared spectroscopy (FTIR) allows rapid non-invasive assessment of biomolecular structures in tissues. In this study, IR-spectra taken from different layers of the pulmonary artery trunk and leaflet tissues of decellularized porcine heart valves were compared with those of pure collagen and elastin, the main protein components in these tissues. In addition, spectral changes associated with aging and oxidative damage were investigated. Infrared absorbance spectra of the arteria intima and media layer were found to be very similar, whereas distinct differences were observed when compared with spectra of the externa layer. In the latter, the shape of the CH-stretching vibration region (3050-2800 cm-1) resembled that of pure collagen. Also, pronounced νCOOH and amide-II bands and a relatively high content of α-helical structures in the externa layer indicated the presence of collagen in this layer. The externa layer of the artery appeared to be sensitive to collagenase treatment, whereas the media and intima layer were particularly affected by elastase and not by collagenase treatment. Protein conformational changes after treatment with collagenase were observed in all three layers. Collagenase treatment completely degraded the leaflet tissue sections. Spectra were also collected from scaffolds after 2 and 12 weeks storage at 37 °C, and after induced oxidative damage. Spectral changes related to aging and oxidative damage were particularly evident in the CH-stretching region, whereas the shape of the amide-I band, reflecting the overall protein secondary structure, remained unaltered.

Raman spectroscopy imaging reveals interplay between atherosclerosis and medial calcification in the human aorta.

Medial calcification in the human aorta accumulates during aging and is known to be aggravated in several diseases. Atherosclerosis, another major cause of cardiovascular calcification, shares some common aggravators. However, the mechanisms of cardiovascular calcification remain poorly understood. To elucidate the relationship between medial aortic calcification and atherosclerosis, we characterized the cross-sectional distributions of the predominant minerals in aortic tissue, apatite and whitlockite, and the associated extracellular matrix. We also compared the cellular changes between atherosclerotic and nonatherosclerotic human aortic tissues. This was achieved through the development of Raman spectroscopy imaging methods that adapted algorithms to distinguish between the major biomolecules present within these tissues. We present a relationship between apatite, cholesterol, and triglyceride in atherosclerosis, with the relative amount of all molecules concurrently increased in the atherosclerotic plaque. Further, the increase in apatite was disproportionately large in relation to whitlockite in the aortic media directly underlying a plaque, indicating that apatite is more pathologically significant in atherosclerosis-aggravated medial calcification. We also discovered a reduction of ß-carotene in the whole aortic intima, including a plaque in atherosclerotic aortic tissues compared to nonatherosclerotic tissues. This unprecedented biomolecular characterization of the aortic tissue furthers our understanding of pathological and physiological cardiovascular calcification events in humans.

Application of acellular intima from porcine thoracic aorta in full-thickness skin wound healing in a rat model.

The tunica intima of aorta is made up of one layer of smooth endothelium and basement membrane. The basement membrane is rich in extracellular matrix (ECM) molecules, including collagen, glycosaminoglycans (e.g., heparan sulfate), proteoglycans (e.g., perlecan), and glycoproteins. All or most of these components are involved in wound healing process. In this work, we determined whether the acellular intima from porcine thoracic aorta can be a new kind of xenograft to repair the skin-wound surface in a rat model. Acellular intima xenografts (AIX) were prepared from tunica intima, and then the swelling ratio, moisture content ratio, water retention ratio, degradation rate, and water vapor transmission rate of the materials were measured. Prothrombin time test was applied to assess its hemostatic property in vitro, in vitro cell experiment was used to test its cellular biocompatibility, and animal experiment was used to evaluate its effect on wound healing. Results showed that AIX, with some reasonable treatments, has good hemostatic property, cell biocompatibility, and histocompatibility. AIX can also promote angiogenesis in the healing process and thus accelerate comprehensive healing, thereby confirming supporting its functionality and excellent application potential in wound healing.

Human pericoronary adipose tissue as storage and possible supply site for oxidized low-density lipoprotein and high-density lipoprotein in coronary artery.

BACKGROUND: Thickening of the pericoronary adipose tissue (PCAT) is a proven risk factor for coronary artery disease, but it is poorly understood whether PCAT stores pro-atherogenic substances with oxidized low-density lipoprotein (oxLDL) and low-density lipoprotein (LDL), and an anti-atherogenic substance with high-density lipoprotein (HDL) and supply them to the coronary intima. METHODS: Using immunohistochemical techniques, the localization of oxLDL, LDL and HDL in PCAT and its adjacent coronary segments was examined in 30 epicardial coronary arteries excised from 11 human autopsy cases. RESULTS: PCAT stored oxLDL and HDL in all, but LDL rarely, in 77 specimens examined, irrespective of the presence or absence of coronary plaques and underlying disease. The percentage (%) incidence of oxLDL, HDL and LDL deposits in intima was, respectively, 28, 10, 35 in 29 normal segments, 80 (p<0.05 vs. normal segments), 12, 75 in 19 white plaques (growth stage), 57, 36, 90 in 15 yellow plaques without necrotic core (NC; mature stage), and 40, 21, 100 (p<0.05 vs. normal segments) in 14 yellow plaques with NC (end-stage of maturation) as classified by angioscopy and histology. In coronary intima, oxLDL deposited in either a dotted or diffuse pattern whereas HDL and LDL showed diffuse patterns. Dotted oxLDL deposits were contained in CD68(+)-macrophages traversing the border of PCAT and adventitia, external and internal elastic laminae. Diffuse oxLDL and HDL deposits colocalized with intimal vasa vasorum. CONCLUSIONS: The results suggested that, as a hitherto unrecognized supplying route, the human PCAT stores oxLDL and HDL and oxLDL is supplied to coronary intima either by CD68(+)-macrophages or vasa vasorum and HDL by vasa vasorum, and that deposition of oxLDL and HDL in the intima increased with plaque growth but the former decreased while the latter increased further with plaque maturation. Molecular therapy targeting PCAT before plaque maturation could be effective in preventing atherosclerosis.

Acellular Endocardium as a Novel Biomaterial for the Intima of Tissue-Engineered Small-Caliber Vascular Grafts.

We aimed to investigate whether acellular endocardium can be used as a useful biomaterial for the intima of engineered small-caliber vascular grafts. Fresh endocardium was harvested from the swine left atrium and was decellularized by digestion with the decellularization solution of Triton X-100 and SDS containing DNase I and RNase A. Surface morphological characteristics and Young's modulus were evaluated. To analyze the effect of mechanical characteristics on cell adhesion, the decellularized endocardium was stiffened with 2.5% glutaraldehyde. Small-caliber vascular grafts were constructed using decellularized endocardium treated with or without glutaraldehyde as the intima. CD34+ cells were seeded onto the luminal surface of the vascular grafts and linked to bioreactors that simulate a pulsatile blood stream. Acellular endocardium had distinct surface morphological characteristics, which were quite different from those of other materials. The compliance of acellular endocardium was higher than that of other materials tested by Young's modulus. CD34+ cells formed a monolayer structure and adhered to the inner face of the acellular endocardium. The glutaraldehyde treatment stiffened the acellular endocardium but had little impact on the surface morphological characteristics or static adhesiveness of the cells. Data from the bioreactor study showed that the detachment of the cells from the surface of glutaraldehyde-treated acellular endocardium increased dramatically when the pressure was equal or higher than 40 mm Hg, while the cells on the untreated acellular endocardium remained well and formed confluent monolayers and tight junctions under the same pressure. Acellular endocardium has distinct structures and mechanical characteristics that are beneficial for CD34+ cell adhesion and retention under dynamic fluid perfusion. Thus, it can be used as a useful biomaterial for the construction of the intima of engineered small-caliber vascular grafts.

Bioinspired One-Dimensional Nano-Wrinkles Guide Liquid Behaviors at the Liquid-Solid Interfaces.

Learning from nature concerning how nanostructured surfaces interact with liquids may provide insight into better understanding of inside living biological interfaces bearing these nanostructures and further development of innovative materials contacting water. Here we investigate the dynamic behaviour of water droplet interacting with one-dimensional nano-wrinkles of different size on polydimethylsiloxane (PDMS) surface. The structure design of the variationally one-dimensional nano-wrinkles is inspired by in vivo responding topographic changes in aortic intima, which was characterized with liquid-phase atomic force microscopy. We show here that increasing the amplitude of the wrinkles promotes the spreading and energy dissipation of liquid droplets on the wrinkled interfaces. This result suggests a possible bio-protection mechanism of blood vessels via its structural changes on the aortic intima against elevated flowing blood, and provides a basis for tuning interfacial nanostructure of optimal durability against wearing by the liquid behaviors.

Intimal sarcoma of the abdominal aorta and common iliac arteries presenting as epithelioid angiosarcoma of the skin: a case report.

Intimal sarcoma (IS) is the most common type of sarcoma of the aorta. IS tumor emboli can involve various organs, including the skin. However, a limited number of IS cases with an initial presentation of skin metastasis has been reported. Cutaneous metastasis as a form of epithelioid angiosarcoma (EAS) has not been well described. Herein, we present a 61-year-old Japanese man with an initial presentation of EAS of the skin, followed by multiple metastases to the skin as a form of EAS prior to detection of IS of the infrarenal aorta and common iliac arteries. In our case, the IS was CD31 and cytokeratin positive but did not express CD34 and factor VIII-related antigen. The EASs in our case exhibited diffuse CD31 expression, and focal factor VIII-related antigen and cytokeratin expression were observed throughout the tumor, including the neoplastic vascular structure; CD34 expression was not identifiable. IS metastasis to the skin has been documented as a form of angiosarcoma. However, IS metastasis has not been well described as a form of EAS. Our case could prove a morphological change from IS to EAS. Given the rarity of primary cutaneous EAS, it is recommended that primary sites other than the skin should be thoroughly investigated when EAS of the skin is encountered.

Fabrication and Short-Term in Vivo Performance of a Natural Elastic Lamina-Polymeric Hybrid Vascular Graft.

Although significant advances have been made in the development of artificial vascular grafts, small-diameter grafts still suffer from excessive platelet activation, thrombus formation, smooth muscle cell intimal hyperplasia, and high occurrences of restenosis. Recent discoveries demonstrating the excellent blood-contacting properties of the natural elastic lamina have raised the possibility that an acellular elastic lamina could effectively serve as a patent blood-contacting surface in engineered vascular grafts. However, the elastic lamina alone lacks the requisite mechanical properties to function as a viable vascular graft. Here, we have screened a wide range of biodegradable and biostable medical-grade polymers for their ability to adhere to the outer surface of the elastic lamina and allow cellular repopulation following engraftment in the rat abdominal aorta. We demonstrate a novel method for the fabrication of elastic lamina-polymeric hybrid small-diameter vascular grafts and identify poly(ether urethane) (PEU 1074A) as ideal for this purpose. In vivo results demonstrate graft patency over 21 days, with low thrombus formation, mild inflammation, and the general absence of smooth muscle cell hyperplasia on the graft's luminal surface. The results provide a new direction for developing small-diameter vascular grafts that are mass-producible, shelf-stable, and universally compatible due to a lack of immune response and inhibit the in-graft restenosis response that is common to nonautologous materials.

Differential expression and regulation of Klotho by paricalcitol in the kidney, parathyroid, and aorta of uremic rats.

Klotho plays an important role in the pathogenesis of cardiovascular disease in chronic kidney disease (CKD). Klotho is highly expressed in the kidney and parathyroid glands, but its presence in the vasculature is debated. Renal Klotho is decreased in CKD, but the effect of uremia on Klotho in other tissues is not defined. The effect of vitamin D receptor activator therapy in CKD on the expression of Klotho in various tissues is also in debate. In uremic rats (surgical 5/6th nephrectomy model), we compared 3 months of treatment with and without paricalcitol on Klotho immunostaining in the kidney, parathyroid glands, and aorta. With uremia, Klotho was unchanged in the parathyroid, significantly decreased in the kidney (66%) and the intimal-medial area of the aorta (69%), and significantly increased in the adventitial area of the aorta (67%) compared with controls. Paricalcitol prevented the decrease of Klotho in the kidney, increased expression in the parathyroid (31%), had no effect in the aortic media, but blunted the increase of Klotho in the aortic adventitia. We propose that fibroblasts are responsible for the expression of Klotho in the adventitia. In hyperplastic human parathyroid tissue from uremic patients, Klotho was higher in oxyphil compared with chief cells. Thus, under our conditions of moderate CKD and mild-to-moderate hyperphosphatemia in rats, the differential expression of Klotho and its regulation by paricalcitol in uremia is tissue-dependent.

Clinical significance of preexisting microcalcification in the iliac artery in renal transplant recipients.

BACKGROUND: The clinical significance of preexisting microcalcification in the iliac artery is undetermined in renal transplant recipients. METHODS: We obtained iliac artery segments from 90 transplant recipients at the time of renal transplantation and performed von Kossa staining for microcalcification. The clinical significance of intimal microcalcification was evaluated with allograft survival rate, rate of graft function decline, and composite of any cardiovascular event or patient death. Expression of fetuin-A and C-reactive protein, key regulators of calcification, was also investigated in the iliac artery. RESULTS: Intimal microcalcification was positive in 48 (53.3%) patients, and its intensity was correlated positively with intimal C-reactive protein intensity (P = 0.019). Allograft survival in patients positive for intimal microcalcification was lower than patients who were negative (P = 0.017). The patients with positivity for both intimal microcalcification and fetuin-A showed lower allograft survival rate than patients with intimal microcalcification positivity alone (P = 0.012). The rate of renal graft function decline was significantly steeper in patients positive for intimal microcalcification than in patients who were negative (P = 0.036). In multivariate analysis, positivity for both intimal microcalcification and fetuin-A was an independent predictor for renal graft function decline (ß = -10.21; P = 0.011). The intimal microcalcification was not associated with composite-event free survival. CONCLUSION: Preexisting intimal microcalcification in the iliac artery predicts a lower allograft survival rate and rapid decline of allograft function. Positivity of fetuin-A with intimal microcalcification further reduces allograft survival rate and an independent predictor for renal graft function decline.

Secondary structure of proteins analyzed ex vivo in vascular wall in diabetic animals using FT-IR spectroscopy.

In recent years many methods for ex vivo tissue analysis or diagnosis of diseases have been applied, including infrared absorption spectroscopy. Fourier-transform infrared (FT-IR) absorption microspectroscopy allows the simultaneous monitoring of the content of various chemical compounds in tissues with both high selectivity and resolution. Imaging of tissue samples in very short time can be performed using a spectrometer equipped with a Focal Plane Array (FPA) detector. Additionally, a detection of minor components or subtle changes associated with the functional status of a tissue sample is possible when advanced methods of data analysis, such as chemometric techniques, are applied. Monitoring of secondary structures of proteins has already proved to be useful in the analysis of animal tissues in disease states. The aim of this work was to build a mathematical model based on FT-IR measurements for the prediction of alterations in the content of secondary structures of proteins analyzed by FT-IR in the vascular wall of diabetic animals. For that purpose a spectral database of proteins of known crystallography and secondary structures was assembled. Thirty-seven proteins were measured by means of two FT-IR techniques: transflection and Attenuated Total Reflectance (ATR). The obtained model was tested on cross-sections of rat tail, for which the content of proteins and their secondary structures was well characterized. Then, the model was applied for the detection of possible alterations in the secondary structures of proteins in the vascular wall of diabetic rats and mice. The obtained results suggest a prominent increase in E- and S-structures and a decrease in the content of H-structures in the vascular wall from diabetic mice and rats. FT-IR-based studies of secondary structures of proteins may be a novel approach to study complex processes ongoing in the vascular wall. The obtained results are satisfactory; however, the existing limitations of the method are also discussed.

Fiber micro-architecture in the longitudinal-radial and circumferential-radial planes of ascending thoracic aortic aneurysm media.

It was recently demonstrated by our group that the delamination strength of ascending thoracic aortic aneurysms (ATAA) was lower than that of control (CTRL, non-aneurysmal) ascending thoracic aorta (ATA), and the reduced strength was more pronounced among bicuspid (BAV) vs. tricuspid aortic valve (TAV) patients, suggesting a different risk of aortic dissection for BAV patients. We hypothesized that aortic valve morphologic phenotype predicts fiber micro-architectural anomalies in ATA. To test the hypothesis, we characterized the micro-architecture in the longitudinal-radial (Z-RAD) and circumferential-radial (Θ-RAD) planes of human ATA tissue that was artificially dissected medially. The outer and inner-media of CTRL-ATA, BAV-ATAA and TAV-ATAA were imaged using multi-photon microscopy in the Z-RAD and Θ-RAD planes to observe collagen and elastin. Micrographs were processed using an image-based tool to quantify several micro-architectural characteristics. In the outer-media of BAV-ATAA, elastin was more undulated and less aligned about the Θ-axis when compared with CTRL-ATA, which is consistent with increased tensile stretch at inflection point of Θ-strips of adventitial-medial half of BAV-ATAA (1.28) when compared with CTRL-ATA (1.13). With increasing age, collagen became more undulated about the Z-axis within the outer-media of TAV-ATAA, and elastin became more oriented in the Z-axis and collagen less radially-oriented within the inner-media of TAV-ATAA. This discrepancy in the micro-architecture with fibers in the inner layers being more stretched and with disrupted radially-oriented components than fibers in the outer layers may be associated with the development, progression and vascular remodeling in aneurysms arising in TAV patients.

Contrast-enhanced MR imaging of atherosclerosis using citrate-coated superparamagnetic iron oxide nanoparticles: calcifying microvesicles as imaging target for plaque characterization.

OBJECTIVE: To evaluate the suitability of citrate-coated very small superparamagnetic iron oxide particles (VSOP) as a contrast agent for identifying inflammation in atherosclerotic lesions using magnetic resonance imaging (MRI). METHODS AND RESULTS: VSOP, which have already been evaluated as a blood pool contrast agent for MR angiography in human clinical trials, were investigated in Watanabe heritable hyper-lipidemic rabbits to determine to what extent their accumulation in atherosclerotic lesions is a function of macrophage density and other characteristics of progressive atherosclerotic plaques. In advanced atherosclerotic lesions, a significant MRI signal loss was found within 1 hour after intravenous administration of VSOP at the intended clinical dose of 0.05 mmol Fe/kg. Histological examinations confirmed correlations between the loss of MRI signal in the vessel wall and the presence of Prussian blue-stained iron colocalized with macrophages in the plaque cap, but surprisingly also with calcifying microvesicles at the intimomedial interface. Critical electrolyte magnesium chloride concentration in combination with Alcian blue stain indicates that highly sulfated glycosaminoglycans are a major constituent of these calcifying microvesicles, which may serve as the key molecules for binding VSOP due to their highly complexing properties. CONCLUSION: Calcifying microvesicles and macrophages are the targets for intravenously injected VSOP in atherosclerotic plaques, suggesting that VSOP-enhanced MRI may render clinically relevant information on the composition and inflammatory activity of progressive atherosclerotic lesions at risk of destabilization.

Adventitial lymphatics and atherosclerosis.

Lymphatic vessels are important in reverse cholesterol transport and play a crucial role in regression of atherosclerotic plaque in experimental animal models. Therefore, we attempted to analyze adventitial microcirculation including lymphatic vessels and adventitial macrophages in large human arteries in various stages of atherosclerosis. Eighty-one arterial segments of large arteries (iliac arteries and abdominal aortas) were obtained from deceased organ donors. Lymphatic vessels were identified using anti-LYVE-1 and anti-D2-40/podoplanin immunohistochemical staining. Adventitial blood vessels and macrophages were visualized using anti-CD-31 and anti-CD-68. Intimal thickness was measured under 100x magnification with an Olympus BX 41 light microscope using the visual mode analySIS 3.2 software. Lymphatic vessels were counted in each cross section of the examined arteries, and adventitial blood vessels (CD31+) were counted using the "hot spot" method. Statistical analysis was performed with Statistica 9.1 PL software (StatSoft, Cracow, Poland). Mann-Whitney, F-Cox, Chi-square, and Spearman's correlation tests were performed and the differences were considered significant at p < 0.05. Lymphatic and blood vessels in the adventitia of examined arteries were identified and quantified. Significant positive correlations were found between the number of adventitial lymphatics (LYVE-L +) and intimal thickness (r = 0.37; p < 0.05) as well as with age of the subjects (r = 0.3; p < 0.05). Thus, lymphatic vessels are present in the adventitia of large arteries in humans and the number of adventitial lymphatic vessels increases with progression of atherosclerosis as assessed by intimal thickness.