Normal Vascular Tissue Research Articles

Identification of breast tumors from diathermy smoke by differential ion mobility spectrometry

IntroductionBreast cancer is the most frequent cancer in women worldwide. The primary treatment is breast-conserving surgery or mastectomy with an adequate clearance margin. Diathermy blade is used extensively in breast-conserving surgery. Surgical smoke produced as a side product has cancer-specific molecular features. Differential mobility spectrometry (DMS) is a rapid and affordable technology for analysis of complex gas mixtures. In our study we examined surgical smoke from malignant and benign breast tissue created with a diathermy blade using DMS. Material and methodsPunch biopsies of 4 mm diameter from breast cancer surgical specimens were taken during gross dissection of fresh surgical specimen and placed in a well plate. The measurement system is a custom-built device called automatic tissue analysis system (ATAS) based on a DMS sensor. Each specimen was incised with a diathermy blade and the surgical smoke was analyzed. ResultsWe examined 106 carcinoma samples from 21 malignant breast tumors. Benign samples (n = 198) included macroscopically normal mammary gland (n = 82), adipose tissue (n = 88) and vascular tissue (n = 28). The classification accuracy when comparing malignant samples to all benign samples was 87%. The sensitivity was 80% and the specificity was 90%. The classification accuracy of carcinomas to ductal and lobular was 94%, 47%, respectively. ConclusionsBenign and malignant breast tissue can be identified with ATAS. These results lay foundation for intraoperative margin assessment with DMS from surgical smoke.

Biomimetic soft fibrous hydrogels for contractile and pharmacologically responsive smooth muscle

The ability to assess changes in smooth muscle contractility and pharmacological responsiveness in normal or pathological-relevant vascular tissue environments is critical to enable vascular drug discovery. However, major challenges remain in both capturing the complexity of in vivo vascular remodeling and evaluating cell contractility in complex, tissue-like environments. Herein, we developed a biomimetic fibrous hydrogel with tunable structure, stiffness, and composition to resemble the native vascular tissue environment. This hydrogel platform was further combined with the combinatory protein array technology as well as advanced approaches to measure cell mechanics and contractility, thus permitting evaluation of smooth muscle functions in a variety of tissue-like microenvironments. Our results demonstrated that biomimetic fibrous structure played a dominant role in smooth muscle function, while the presentation of adhesion proteins co-regulated it to various degrees. Specifically, fibre networks enabled cell infiltration and upregulated expression of actomyosin proteins in contrast to flat hydrogels. Remarkably, fibrous structure and physiologically relevant stiffness of hydrogels cooperatively enhanced smooth muscle contractility and pharmacological responses to vasoactive drugs at both the single cell and intact tissue levels. Together, this study is the first to demonstrate alterations of human vascular smooth muscle contractility and pharmacological responsiveness in biomimetic soft, fibrous environments with a cellular array platform. The integrated platform produced here could enable investigations for pathobiology and pharmacological interventions by developing a broad range of patho-physiologically relevant in vitro tissue models. Statement of SignificanceEngineering functional smooth muscle in vitro holds the great potential for diseased tissue replacement and drug testing. A central challenge is recapitulating the smooth muscle contractility and pharmacological responses given its significant phenotypic plasticity in response to changes in environment. We present a biomimetic fibrous hydrogel with tunable structure, stiffness, and composition that enables the creation of functional smooth muscle tissues in the native-like vascular tissue microenvironment. Such fibrous hydrogel is further combined with the combinatory protein array technology to construct a cellular array for evaluation of smooth muscle phenotype, contraction, and cell mechanics. The integrated platform produced here could be promising for developing a broad range of normal or diseased in vitro tissue models.

Analysis of the Expression of Angioarchitecture-related Factors in Patients with Cerebral Arteriovenous Malformation.

Background:Cerebral arteriovenous malformation (cAVM) is a type of vascular malformation associated with vascular remodeling, hemodynamic imbalance, and inflammation. We detected four angioarchitecture-related cytokines to make a better understanding of the potential aberrant signaling in the pathogenesis of cAVM and found useful proteins in predicting the risk of cerebral hemorrhage.Methods:Immunohistochemical analysis was conducted on specimens from twenty patients with cAVM diagnosed via magnetic resonance imaging and digital subtraction angiography and twenty primary epilepsy controls using antibodies against vascular endothelial growth factor receptor-2 (VEGFR-2), matrix metalloproteinase-9 (MMP-9), vascular cell adhesion molecule (VCAM-1), and endothelial nitric oxide synthase (eNOS). Western blotting and real-time fluorescent quantitative polymerase chain reaction (PCR) were performed to determine protein and mRNA expression levels. Student's t-test was used for statistical analysis.Results:VEGFR-2, MMP-9, VCAM-1, and eNOS expression levels increased in patients with cAVM compared with those in normal cerebral vascular tissue, as determined by immunohistochemical analysis. In addition, Western blotting and real-time PCR showed that the protein and mRNA expression levels of VEGFR-2, MMP-9, VCAM-1, and eNOS were higher in the cAVM group than in the control group, all the differences mentioned were statistically significant (P < 0.05).Conclusions:VEGFR-2, MMP-9, VCAM-1, and eNOS are upregulated in patients with cAVM and might play important roles in angiogenesis, vascular remodeling, and migration in patients with cAVM. MMP-9, VEGFR-2, VCAM-1, and eNOS might be potential excellent group proteins in predicting the risk of cerebral hemorrhage at arteriovenous malformation.

The expression levels of TNF-α and IL-6 in tumor tissues and change of serum ferritin concentration in patients with intracranial aneurysm and its clinical significance

Objective To investigate the expression levels of tumor necrosis factor alpha (TNF-α), interleukin-6(IL-6) and the change of serum ferritin in patients with intracranial aneurysm and its clinical significance. Methods 22 patients with intracranial aneurysm and 16 cases of traumatic brain injury who recevied operation in Department of Neurosurgery in Quzhou People's Hospital from Jan.2014 to Jan.2015 were enrolled as observation group and control group.The intracranial aneurysm tissues and normal cerebral vascular tissues were collected respectively to detect the situation of inflammatory cell infiltration, and the expression of TNF-α and IL-6 was detected by HE staining and immunohistochemical staining.The serum was collected on hospitalized, postoperative 3 days and postoperative 7 days to detect serum ferritin. Results The positive cells were observed in the observation group after immunohistochemical staining, in which the expression levels of TNF-α[(0.194±0.074) vs.(0.135±0.047), t=2.799]and IL-6[(0.152±0.057) vs.(0.103±0.028), t=3.494]were significantly higher than those in the control group(P=0.008, 0.001). The serum ferritin level in the observation group was significantly higher than that in the control group at postoperative 3d[(232.25±105.26)ng/mL vs.(169.51±66.24)ng/mL, t=2.097]and postoperative 7d[(263.39±114.73)ng/mL vs.(166.57±73.71)ng/mL, t=2.955](P=0.043, 0.005). The difference of serum ferritin on different day in the observation group also was statistically significant(F=8.625, P=0.003). Conclusion The expression levels of TNF-α and IL-6 in intracranial aneurysms were high, which may be one of the important factors in the formation of intracranial aneurysms, and it provides an important reference of early prevention and drug treatment for patients with intracranial aneurysm.The serum ferritin is related to the rupture and hemorrhage of intracranial aneurysm, which can be used to judge prognosis and guide rational treatment, and worth further study to confirm. Key words: Intracranial aneurysm; Tumor necrosis factor-α; Interleukin-6; Serum ferritin

Propranolol inhibits the proliferation, migration and tube formation of hemangioma cells through HIF-1α dependent mechanisms.

The aim of this study was to investigate the mechanism of propranolol on the regression of hemangiomas. Propranolol-treated hemangioma tissues were collected and the expression of hypoxia inducible factor-1α (HIF-1α) was examined. We also established HIF-1α overexpression and knockdown hemangioma cells, and determined the effects of HIF-1α on the hemangioma cells proliferation, apoptosis, migration and tube formation. Significantly increased HIF-1α level was found in the hemangioma tissues compared to that in normal vascular tissues, whereas propranolol treatment decreased the HIF-1α level in hemangioma tissues in a time- and dose-dependent manner. Moreover, propranolol treatment significantly decreased cell proliferation, migration and tube formation as well as promoted cell apoptosis in HIF-1α overexpression and knockdown hemangioma cells. Propranolol suppressed the cells proliferation, migration and tube formation of hemangioma cells through HIF-1α dependent mechanisms. HIF-1α could serve as a novel target in the treatment of hemangiomas.

MiR-106b-5p Inhibits Tumor Necrosis Factor-α-induced Apoptosis by Targeting Phosphatase and Tensin Homolog Deleted on Chromosome 10 in Vascular Endothelial Cells.

Background:Apoptosis of endothelial cells (ECs) plays a key role in the development of atherosclerosis and there are also evidence indicated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a viable target in therapeutic approaches to prevent vascular ECs apoptosis. Aberrant miR-106b-5p expression has been reported in the plasma of patients with unstable atherosclerotic plaques. However, the role and underlying mechanism of miR-106-5p in the genesis of atherosclerosis have not been addressed. In this study, we explored the anti-apoptotic role of miR-106-5p by regulating PTEN expression in vascular ECs.Methods:Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the expression levels of miR-106b-5p in human atherosclerotic plaques and normal vascular tissues. Human umbilical vein endothelial cells (HUVEC) were transfected with miR-106b-5p mimic or negative control mimic, and apoptosis was induced by serum starvation and tumor necrosis factor-α (TNF-α) treat. Western blotting and real-time RT-PCR experiments were used to detect PTEN expression levels and TNF-α-induced apoptosis was evaluated by the activation of caspase-3 and cell DNA fragmentation levels in HUVEC.Results:The expression of miR-106b-5p was significantly downregulated in plaques than in normal vascular tissues. TNF-α significantly downregulated miR-106b-5p expression levels and upregulated activation of caspase-3 and cell DNA fragmentation levels in HUVEC. Overexpression of miR-106b-5p with miR-106b-5p mimic inhibited PTEN expression and TNF-α-induced apoptosis in HUVEC. Luciferase reporter assays confirmed that miR-106b-5p binds to PTEN mRNA 3’ untranslated region site.Conclusion:MiR-106b-5p could inhibit the expression of PTEN in vascular ECs, which could block TNF-α-induced activation of caspase-3, thus prevent ECs apoptosis in atherosclerosis diseases.

Notch receptor expression in human brain arteriovenous malformations.

The roles of the Notch pathway proteins in normal adult vascular physiology and the pathogenesis of brain arteriovenous malformations are not well-understood. Notch 1 and 4 have been detected in human and mutant mice vascular malformations respectively. Although mutations in the human Notch 3 gene caused a genetic form of vascular stroke and dementia, its role in arteriovenous malformations development has been unknown. In this study, we performed immunohistochemistry screening on tissue microarrays containing eight surgically resected human brain arteriovenous malformations and 10 control surgical epilepsy samples. The tissue microarrays were evaluated for Notch 1–4 expression. We have found that compared to normal brain vascular tissue Notch-3 was dramatically increased in brain arteriovenous malformations. Similarly, Notch 4 labelling was also increased in vascular malformations and was confirmed by western blot analysis. Notch 2 was not detectable in any of the human vessels analysed. Using both immunohistochemistry on microarrays and western blot analysis, we have found that Notch-1 expression was detectable in control vessels, and discovered a significant decrease of Notch 1 expression in vascular malformations. We have demonstrated that Notch 3 and 4, and not Notch 1, were highly increased in human arteriovenous malformations. Our findings suggested that Notch 4, and more importantly, Notch 3, may play a role in the development and pathobiology of human arteriovenous malformations.

Integrated Analysis of Microarray Data of Atherosclerotic Plaques: Modulation of the Ubiquitin-Proteasome System

Atherosclerosis is a typical complex multi-factorial disease and many molecules at different levels and pathways were involved in its development. Some studies have investigated the dysregulation in atherosclerosis at mRNA, miRNA or DNA methylation level, respectively. However, to our knowledge, the studies that integrated these data and revealed the abnormal networks of atherosclerosis have not been reported. Using microarray technology, we analyzed the omics data in atherosclerosis at mRNA, miRNA and DNA methylation levels. Our results demonstrated that the global DNA methylation and expression of miRNA/mRNA were significantly decreased in atherosclerotic plaque than in normal vascular tissue. The interaction network constructed using the integrative data revealed many genes, cellular processes and signaling pathways which were widely considered to play crucial roles in atherosclerosis and also revealed some genes, miRNAs or signaling pathways which have not been investigated in atherosclerosis until now (e.g. miR-519d and SNTB2). Moreover, the overall protein ubiquitination in atherosclerotic plaque was significantly increased. The proteasome activity was increased early but decreased in advanced atherosclerosis. Our study revealed many classic and novel genes and miRNAs involved in atherosclerosis and indicated the effects of ubiquitin-proteasome system on atherosclerosis might be closely related to the course of atherosclerosis. However, the efficacy of proteasome inhibitors in the treatment of atherosclerosis still needs more research.

Abstract 3002: ID3 expression and oxidative DNA damage are associated with growth of benign and malignant vascular lesions

Abstract Higher levels of Id3 expression have been reported in tumor-infiltrating blood vessels from various tumors, but the exact role of Id3 in vascular lesion formation is poorly understood. This study aims at identifying the status of Id3 expression and distribution in benign vascular lesions and attempts to correlate this information with the progression of vascular lesion. In this study we investigated the expression pattern of Id3 in malignant, benign, and normal human vascular tissue specimens. Expression analysis of Id3 was carried out using immunohistochemical analysis in 48 sporadic samples of a heterogeneous group of benign and malignant vascular tissue. Immunohistochemical analysis confirmed that many of the malignant vascular lesions exhibited strong Id3 expression that correlated positively with increased 8-hydroxydeoxyguanosine (8-OHdG) a marker of oxidative DNA damage. In contrast, in normal human vascular tissue, Id3 expression and 8-OHdG was low. In malignant vascular tissue, Id3 protein displayed a stronger nuclear localization when compared to the cytoplasm. Id3 expression levels in benign vascular lesions while still expressing Id3 were to a lesser extent when compared to malignant vascular tissue. To our knowledge, this is the first study to compare benign and malignant vascular lesions to assess the role of Id3 and 8-OHdG. Our data reveals Id3 expression and oxidative DNA damage to be highly associated in malignant cells and to a lesser extent in their benign counterparts, indicating that the growth of vascular lesions may require oxidative stress signaling converging on Id3. The results also support the involvement of Id3 in tumor angiogenesis a process that critically influences the malignant behavior of cancer cells. Citation Format: Quentin H. Felty, Jayanta K. Das. ID3 expression and oxidative DNA damage are associated with growth of benign and malignant vascular lesions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3002. doi:10.1158/1538-7445.AM2014-3002

Inflammation induced-endothelial cells release angiogenesis associated-microRNAs into circulation by microparticles

Background Endothelial cells derived microRNAs can be detected in plasma and serum and there is evidence that inflammatory disease states may affect the levels of circulating microRNAs. However, there is no direct proof that inflammation induces endothelial cells to release microRNAs into circulation. This study aimed to explore whether inflammation could induce endothelial cells to release microRNAs into circulation and to investigate whether these released microRNAs derived from endothelial cells were transported in microparticles. Methods Microparticles were isolated from human atherosclerotic plaques with an active inflammatory phenotype and normal vascular tissue. Flow cytometry and real-time PCR were used to detect the levels of microparticles and microRNAs. Human umbilical vein endothelial cells (HUVEC) was treated with tumour necrosis factor α (TNF-α, 10 ng/ml) for 24 hours, and then HUVEC and the culture medium were respectively collected. Results By comparing microparticles isolated from human atherosclerotic plaques with an active inflammatory phenotype (n=9) and those from normal vascular tissues (n=9), we found levels of annexin V+ microparticles and annexin V+ CD144+ microparticles were significantly increased in plaques and angiogenesis associated microRNAs (106b, 25, 92a and 21) were also significantly increased in microparticles from plaques. After exposure to TNF-α at a concentration of 10 ng/ml (TNF-α group, n=3) or DMEM (control group, n=3) for 24 hours, counts of microparticles and expressions of microRNAs 106b, 25, 92a and 21 in microparticles isolated from medium significantly increased. However, there were no differences in the intracellular levels of microRNAs 25, 92a or 21 isolated from HUVEC between TNF-α group and control group, while microRNA 106b decreased in TNF-α group. Conclusion Inflammation could induce endothelial cells to release angiogenesis associated microRNAs into circulation, causing higher levels of circulating endothelial cells derived microRNAs in atherosclerosis.

Idiopathic congenital true leukonychia totalis.

A 16-year-old male presented with white-colored fingernails since birth [Figure 1]. Toenails were normal. None of the family members were affected. Hematological investigations were normal. On cutaneous examination all finger nails were milky, porcelain white in color and smooth surfaced. No knuckle pads, palmoplantar keratoderma, or hearing loss was observed. General and systemic examination was normal. On clinical correlation, a diagnosis of idiopathic congenital true leukonychia totalis was made. Figure 1 Milky, porcelain, white-colored smooth-surfaced finger nails Leukonychia is the white discoloration of nail that loses its normal pink color, with disappearance of the lunula. It is the commonest chromatic abnormality of the nail apparatus. It is also referred to as white nail, milk spots, and fortune or gift spots. It is derived from the Greek word “leukos” means white and “onyx” means nail.[1,2] Leukonychia is classified as: [Table 1]. Table 1 Classification of leukonychia The proposed white discoloration of the nail is attributable to reflection of light from large keratohyaline granules present in the parakeratotic cells of the ventral portion of nail plate thus preventing the visualisation of normal vascular tissue underlying the nail plate.[2,3] Total congenital leukonychia is very rare hereditary condition in which all nails are milky white with an autosomal dominant inheritance but it can be autosomal recessive in few cases. Hereditary leukonychia involves whole nail plate as against acquired one which shows transverse or punctate leukonychia. Mutations in PLCD1 gene on chromosome 3p21.3-p22 was identified as cause of hereditary leukonychia. This PLCD1 gene is localized to nail matrix and encodes phosphoinositide specific phospholipase C delta 1 subunit which is important enzyme in phosphoinositide metabolism required for molecular control of color and growth of nail.[4] Leukonychia totalis is associated with multiple systemic diseases such as diabetes, liver cirrhosis, renal failure, cardiac failure, gall stones, renal calculi, duodenal ulcers, pili torti, trichilemmal cyst, sebaceous cyst, congenital hyperparathyroidism, Hodgkin's lymphoma, Leopard syndrome, epiphyseal dysplasia syndrome, Bart Pumphrey syndrome, mental retardation, etc., However, there are very few reports in the literature on sporadic congenital leukonychia totalis without any systemic abnormalities.[1,3] Treatment is usually supportive with detailed investigation to check for association of any systemic or genetic disorders.

Factors affecting the selectivity of nanoparticle-based photoinduced damage in free and xenografted chorioallantoïc membrane model

Background: Photodynamic therapy (PDT) is a minimally invasive treatment modality for selective destruction of tumours. Critical anatomical structures, like blood vessels in close proximity to the tumour, could be harmed during PDT.Purpose: This study aims to discriminate the photoinduced response of normal and cancerous tissues to photodamage induced by liposomal formulations of meta-tetra(hydroxyphenyl)chlorin (mTHPC).Methods: Normal vascular and cancerous tissues were represented, respectively, by free and xenografted in vivo model of chick chorioallantoïc membrane (CAM). Eggs received an intravenous administration of plain (Foslip®) or stabilised formulations (Fospeg®). Drug release and liposome destruction were, respectively, determined by photoinduced quenching and nanoparticle tracking analysis. PDT was performed at different drug-light intervals (DLI) with further assessment of photothrombic activity, tumoritropism and photoinduced necrosis.Results: Compared to Foslip®, Fospeg® demonstrated significantly higher stability, slower drug release, better tumoricidal effect and lower damage to the normal vasculature at already 1 h DLI.Discussion: This work suggests that nanoparticle-based PDT selectivity could be optimised by analyzing the photoinduced damage of healthy and tumour tissues.Conclusion: In fine, Fospeg® appeared to be the ideal candidate in clinical context due to its potential to destroy tumours and reduce vascular damage to normal tissues at short DLI.

Notch4 is activated in endothelial and smooth muscle cells in human brain arteriovenous malformations

Up-regulation of Notch4 was observed in the endothelial cells in the arteriovenous malformations (AVMs) in mice. However, whether Notch4 is also involved in brain AVMs in humans remains unclear. Here, we performed immunohistochemistry on normal brain vascular tissue and surgically resected brain AVMs and found that Notch4 was up-regulated in the subset of abnormal vessels of the brain AVM nidus, compared with control brain vascular tissue. Two-photon confocal images show that Notch4 was expressed not only in the endothelial but also in the smooth muscle cells of the vascular wall in brain AVMs. Western blotting shows that Notch4 was activated in brain AVMs, but not in middle cerebral artery of normal human brain, which was confirmed by immunostaining. Our findings suggest a possible contribution of Notch4 signalling to the development of brain AVMs in human.

Degradome Products of the Matricellular Protein CCN1 as Modulators of Pathological Angiogenesis in the Retina

CCN1 is a matricellular protein involved in normal vascular development and tissue repair. CCN1 exhibits cell- and context-dependent activities that are reflective of its tetramodular structure phylogenetically linked to four domains found in various matrix proteins. Here, we show that vitreal fluids from patients with proliferative diabetic retinopathy (PDR) were enriched with a two-module form of CCN1 comprising completely or partially the insulin-like growth factor-binding protein (IGFBP) and von Willebrand factor type C (vWC) domains. The two- and three-module forms comprising, in addition to IGFBP and vWC, the thrombospondin type 1 (TSP1) repeats are CCN1 degradome products by matrix metalloproteinase-2 and -14. The functional significance of CCN1 and its truncated variants was determined in the mouse model of oxygen-induced retinopathy, which simulates neovascular growth associated with PDR and assesses treatment outcomes. In this model, lentivirus-mediated expression of either CCN1 or the IGFBP-vWC-TSP1 form reduced ischemia-induced neovascularization, whereas ectopic expression of the IGFBP-vWC variant exacerbated pathological angiogenesis. The IGFBP-vWC form has potent proangiogenic properties promoting retinal endothelial cell growth, migration, and three-dimensional tubular structure formation, whereas the IGFBP-vWC-TSP1 variant suppressed cell growth and angiogenic gene expression. Both IGFBP-vWC and IGFBP-vWC-TSP1 forms exhibited predictable variations of their domain folding that enhanced their functional potential. These data provide new insights into the formation and activities of CCN1-truncated variants and raise the predictive value of the form containing completely or partially the IGFBP and vWC domains as a surrogate marker of CCN1 activity in PDR distinguishing pathological from physiological angiogenesis.

High-resolution vascular tissue characterization in mice using 55 MHz ultrasound hybrid imaging

Ultrasound and Duplex ultrasonography in particular are routinely used to diagnose cardiovascular disease (CVD), which is the leading cause of morbidity and mortality worldwide. However, these techniques may not be able to characterize vascular tissue compositional changes due to CVD. This work describes an ultrasound-based hybrid imaging technique that can be used for vascular tissue characterization and the diagnosis of atherosclerosis. Ultrasound radiofrequency (RF) data were acquired and processed in time, frequency, and wavelet domains to extract six parameters including time integrated backscatter (TIB), time variance (Tvar), time entropy (TE), frequency integrated backscatter (FIB), wavelet root mean square value (Wrms), and wavelet integrated backscatter (WIB). Each parameter was used to reconstruct an image co-registered to morphological B-scan. The combined set of hybrid images were used to characterize vascular tissue in vitro and in vivo using three mouse models including control (C57BL/6), and atherosclerotic apolipoprotein E-knockout (APOE-KO) and APOE/A1 adenosine receptor double knockout (DKO) mice. The technique was tested using high-frequency ultrasound including single-element (center frequency=55MHz) and commercial array (center frequency=40MHz) systems providing superior spatial resolutions of 24μm and 40μm, respectively. Atherosclerotic vascular lesions in the APOE-KO mouse exhibited the highest values (contrast) of −10.11±1.92dB, −12.13±2.13dB, −7.54±1.45dB, −5.10±1.06dB, −5.25±0.94dB, and −10.23±2.12dB in TIB, Tvar, TE, FIB, Wrms, WIB hybrid images (n=10, p<0.05), respectively. Control segments of normal vascular tissue showed the lowest values of −20.20±2.71dB, −22.54±4.54dB, −14.94±2.05dB, −9.64±1.34dB, −10.20±1.27dB, and −19.36±3.24dB in same hybrid images (n=6, p<0.05). Results from both histology and optical images showed good agreement with ultrasound findings within a maximum error of 3.6% in lesion estimation. This study demonstrated the feasibility of a high-resolution hybrid imaging technique to diagnose atherosclerosis and characterize plaque components in mouse. In the future, it can be easily implemented on commercial ultrasound systems and eventually translated into clinics as a screening tool for atherosclerosis and the assessment of vulnerable plaques.

The Chromosome 9p21.3 Coronary Heart Disease Risk Allele Is Associated with Altered Gene Expression in Normal Heart and Vascular Tissues

Genome-wide association studies have identified a coronary artery disease (CAD) risk locus in a non-coding region at 9p21.3, the nearest genes being CDKN2A and CDKN2B. To understand the pathways by which this locus might influence CAD susceptibility, we investigated associations between the 9p21.3 risk genotype and global gene expression in heart tissue from donors with no diagnosed heart disease (n = 108, predominant cause of death, cerebral vascular accident) and in carotid plaque (n = 106), aorta (n = 104) and mammary artery (n = 88) tissues from heart valve and carotid endarterectomy patients. Genotyping was performed with Taqman assays and Illumina arrays, and gene expression profiles generated with Affymetrix microarrays. Associations were analyzed with an additive genetic model. In heart tissue, 46 genes were putatively altered in association with the 9p21.3 risk allele (70% down-regulated, fold-change >1.1 per allele, p<0.05 adjusted for age, gender, ethnicity, cause of death). These genes were enriched for biomarkers of myocardial infarction (p = 1.53×10−9), response to wounding (p = 2.65×10−10) and inflammatory processes (p<1.97×10−7). Among the top 10 most down-regulated genes, 7 genes shared a set of transcription factor binding sites within conserved promoter regions (p<1.14×10−5), suggesting they may be co-regulated. Canonical pathway modelling of the most differentially expressed transcripts across all tissues (154 genes, 60% down-regulated, fold-change >1.1 per allele, p<0.01) showed that 75% of the genes could be transcriptionally regulated through the cell cycle G1 phase progression pathway (p<1.08×10−258), in which CDKN2A and CDKN2B play a regulatory role. These data suggest that the cell cycle G1 phase progression pathway is activated in individuals with the 9p21.3 risk allele. This may contribute to a proliferative phenotype that promotes adverse cardiac hypertrophy and vascular remodeling, leading to an increased CAD risk.

An in vitro reconstitution system to address the mechanism of the vascular expression of the bradykinin B1 receptor in response to angiotensin converting enzyme inhibition

The expression of the bradykinin (BK) B1 receptor (B1R), lacking in normal vascular tissues, is induced following innate immune system activation and chronic blockade of angiotensin converting enzyme (ACE). To identify cytokine-dependent or -independent mechanisms for the latter phenomenon, the ACE inhibitor enalaprilat and several peptides potentiated in vivo by ACE blockade were applied either directly to human umbilical artery smooth muscle cells (hUA-SMCs) or to differentiated monoblastoid U937 cells to produce a conditioned medium (CM) that was later transferred to hUA-SMCs. A phagocyte stimulant, lipopolysaccharide, did not upregulate B1R, measured using [3H]Lys-des-Arg9-BK binding, or translocate NF-κB to the nuclei if applied directly to the hUA-SMCs. However, the CM of lipopolysaccharide-stimulated U937 cells was active in these respects (effects inhibited by etanercept and correlated to TNF-α presence in the CM). A peptidase-resistant B1R agonist had no significant direct or indirect acute effect (4h) on B1R expression, but repeated hUA-SMC stimulations over 40h were stimulatory in the absence of NF-κB activation. Other peptides regulated by ACE or enalaprilat did not directly or indirectly stimulate B1R expression. The reconstitution system supports the rapid cytokine-dependent vascular induction of B1Rs and a slow “autoregulatory” one potentially relevant for the ACE blockade effect.

Autofluorescence manifestation in related lesions of peripheral retinopathy

Objective To observe the autofluorescence (AF) manifestation in related lesions of periphery retinopathy. Methods Sixty eyes of 42 patients with periphery retinopathy underwent the examination of Optomap fundus photograph (200°) and fundus fluorescein angiography (FFA). The HRA Ⅱ melanin-related near-infrared fundus autofluorescence (NIA, excitation 795 nm) and lipofuscin-related fundus autofluorescence (FAF, excitation 488 nm) were measured for all the patients. The AF was recorded with nine images per second, and then a final AF image with 55° view and 822 × 768 pixel was generated by the HRA. AF images can be valuable or valueless if there was or was not visible blood vessels and related retinal tissues on the image. AF from lesion regions can be normal or abnormal fluorescence comparing to the normal vascular and retinal tissue AF. The abnormal fluorescence was divided into no AF, weak AF and strong AF relative to the background grayscale. The grading consistency of abnormal fluorescence based on FAF and NIA examination was comparatively analyzed. Results Valuable AF images were captured in 53/60 eyes (88. 33%)and valueless AF images were captured in 7/60 eyes (11.67%). Among 53 eyes with valuable AF image, NIA showed normal fluorescence in 28 eyes (52. 83%), abnormal fluorescence with sheet-like, dot-shaped or stripped in 25 eyes (47.17%); FAF showed normal fluorescence in two eyes (3.77 % ), abnormal fluorescence with sheet-like, scattered along vessels or pigments in 51 eyes (96.23 % ).Twenty-five eyes with abnormal fluorescence were observed both in two examinations, including same grades in 18 eye (72.00%) and different grades in seven eyes (28.00%). Conclusion The AF manifestation with different levels exists in related lesions of periphery retinopathy. Key words: Retinal pigments/diagnostic use; Lipofuscin/analysis; Microscopy, confocal/methods; Image processing, computer-assisted; Pigment epithelium of eye/metabolism

EBP50 inhibits the anti-mitogenic action of the parathyroid hormone type 1 receptor in vascular smooth muscle cells

Parathyroid hormone-related protein (PTHrP) and the parathyroid hormone type 1 receptor (PTH1R) are important regulators of vascular remodeling. PTHrP expression is associated to increased proliferation of vascular smooth muscle cells (VSMC). In contrast, signaling via the PTH1R inhibits cell growth. The mechanisms regulating the dual effect of PTHrP and PTH1R on VSMC proliferation are only partially understood. In this study we examined the role of the adaptor protein ezrin–radixin–moesin-binding phosphoprotein (EBP50) on PTH1R expression, trafficking, signaling and control of A10 cell proliferation. In normal rat vascular tissues, EBP50 was restricted to the endothelium with little expression in VSMC. EBP50 expression significantly increased in VSMC following angioplasty in parallel with PTHrP. Interestingly, PTHrP was able to induce EBP50 expression. In the clonal rat aortic smooth muscle cell line A10, EBP50 increased the recruitment of PTH1R to the cell membrane and delayed its internalization in response to PTHrP(1–36). This effect required an intact C-terminal motif in the PTH1R. In naïve A10 cells, PTHrP(1–36) stimulated cAMP production but not intracellular calcium release. In contrast, PTHrP(1–36) induced both cAMP and calcium signaling in A10 cells over-expressing EBP50. Finally, EBP50 attenuated the induction of p27 kip1 and the anti-proliferative effect of PTHrP(1–36). In summary, this study demonstrates the dynamic expression of EBP50 in vessels following injury and the effects of EBP50 on PTH1R function in VSMC. These findings highlight one of the mechanisms leading to increased VSMC proliferation and have important implication in the understanding of the molecular events leading to restenosis.

837 Concomitant targeting of cyclooxygenase-2 and oxidant stress pathways for radioprotection of normal vascular tissue

Ephrin receptors (Eph) affect cell shape and movement, unlike other receptor tyrosine kinases that directly affect proliferative pathways. The kinase domain of EphA3 is activated by ephrin binding and receptor oligomerization. This activation is associated with two tyrosines in the juxtamembrane region; these tyrosines are sites of autophosphorylation and interact with the active site of the kinase to modulate activity. This allosteric event has important implications both in terms of understanding signal transduction pathways mediated by Eph kinases as well as discovering specific therapeutic ligands for receptor kinases. In order to provide further details of the molecular mechanism through which the unphosphorylated juxtamemebrane region blocks catalysis, we studied wild-type and site-specific mutants in detail. High-resolution structures of multiple states of EphA3 kinase with and without the juxtamembrane segment allowed us to map the coupled pathway of residues that connect the juxtamembrane segment, the activation loop, and the catalytic residues of the kinase domain. This highly conserved set of residues likely delineates a molecular recognition pathway for most of the Eph RTKs, helping to characterize the dynamic nature of these physiologically important enzymes.