Osteoglycin expression and localization in rabbit tissues and atherosclerotic plaques

The localization of osteoglycin (OG), one of the corneal keratan sulfate proteoglycans, was studied in different normal rabbit tissues, as well as in atherosclerotic lesions, by means of in situ hybridization and immunohistochemistry. OG was associated with the vasculature of all the organs analyzed. Normal aortas showed abundance of the protein in the adventitia and focally in the media. Peripheral vessels showed OG localized only in the adventitia. OG mRNA was restricted to vascular smooth muscle cells, pericytes, and fibroblasts in aorta and skeletal muscle. In striated muscle, OG was abundant and distributed in foci around muscles and vessels, whereas in visceral muscle, the protein was homogeneously distributed throughout the extracellular matrix. In all the other organs studied, OG was only associated with the vasculature, with the exception of the lung and liver. In these two organs, the protein accumulated also around cartilage, alveoli, and hepatic duct. In atherosclerotic lesions, OG mRNA was down-regulated in the media and up-regulated in the activated endothelium and thick neo-intima, whereas the protein accumulated in the front edge of migrating smooth muscle cells. We conclude that OG is a basic component of the vascular extracellular matrix. OG also plays a role in atherosclerosis, and might be useful for therapeutic interventions. In addition, the possible involvement of OG in maintaining physical properties of tissues is discussed.     

Embryonic stem cells markers are present within rabbit atherosclerotic plaques

Recent evidence suggests that smooth muscle cells within atherosclerotic plaques originate from vascular progenitor cells. We have previously shown that smooth muscle cells and macrophages present within rabbit atherosclerotic plaques are positive for factors of the renin angiotensin and nitric oxide systems as well as the hematopoietic stem-cell marker CD34 and the pan-leukocyte marker CD45. To explore the idea that these cells are of primitive types, immunohistochemistry was used to identify pluripotent embryonic stem cells (ESC) markers (Oct-4, SSEA1,3,4, TRA1-60, 81) in these plaques and to compare these to intimal thickening. Objective: To immunolocalise ESC markers in rabbit aortic intimal thickening and atherosclerotic plaques. Design: New Zealand White rabbits were fed either a control (Con) diet, 0.5% cholesterol (Chol) or 1% methionine (Meth) for 12 weeks. Animals were perfusion fixed, aortae excised and processed for paraffin. Immunohistochemistry was performed by standard techniques. Results: Oct-4, SSEA 1, 3 and 4, TRA-1-60 and TRA-1-81 were all present within in atherosclerotic plaques. However, some cells were not positive for TRA-1-60 and TRA-1-81. In fact, positive TRA-1-81 macrophages were uncommon, and positive TRA-1-81 smooth muscle cells were rare. Intimal thickening in Meth did not show any TRA-1-81 positive cells Conclusions: Macrophages and smooth muscle cells within atherosclerotic plaques express markers of ESC. These results suggest that cells within these plaques are primitive and might differentiate into other types of cells.     

Apoptosis in human atherosclerotic plaques

Intimal cell death has been a recognized feature of advanced atherosclerotic disease. With the advent of DNA in situ end labelling and/or ultrastructural techniques, recent findings suggest that cells of an atheroma undergo programmed cell death or apoptosis. The pathophysiologic relevance of apoptosis in atherosclerotic disease is debatable. Apoptotic cell death may influence lesion progression and thus reduce overall plaque burden. Alternatively, apoptosis may prove a means of quenching the inflammation, converting cellular-rich lesions to so-called stable fibrous hypocellular plaques or conversely weaken the fibrous cap causing plaque rupture, a major cause of acute coronary syndromes. Apoptotic cells within plaques are typically macrophages, smooth muscle cells and T-cells and the frequency of death varies in the different regions of the lesion. The precise signalling pathways of apoptosis in plaques are unknown. There is however, some evidence that production of immune cytokines may promote apoptosis through activation of the Fas ligand-mediated death pathway. Genetic signals that regulate apoptosis in the atheroma, at least in smooth muscle cells, may involve the tumour suppressor genes p105 ^RB and p53. Further studies as to the relevance of apoptosis in acute coronary syndromes and potential mechanisms are emerging.     

Correlations between gene expression highlight a different activation of ACE/TLR4/PTGS2 signaling in symptomatic and asymptomatic plaques in atherosclerotic patients

Molecular Biology Reports - Inflammation has a key role and translates the effects of many known risk factors for the disease in atherosclerotic vulnerable plaques. Aiming to look into the elements...     

Methylation profiling of human atherosclerotic plaques

Somatic mutation theory of atherogenesis proved by alterations at the DNA level such as "loss of heterozygosity" and microsatellite instability in atherosclerotic plaque is complemented by the date of epigenetic variability of genetic loci involved in the pathological process. However, only recently large-scale analysis of epigenetic modifications in the human genome became possible. For the first time quantitative microarray-based methylation profiling of 1505 CpG-sites across 807 genes was performed in atherosclerotic aorta and carotid artery wall lesions using the GoldenGate Methylation Cancer Panel I ("Illumina", USA). One hundred and three (7%) CpG-sites in 90 (11%) genes were differentially methylated between tissue samples. The most pronounced differences in DNA methylation levels were registered for a site which is located in CpG-island of imprinted gene H19. By comparing 90 genes that were differentially methylated between tissue samples in our study, 10 genes (ICAM1, GSTM1, IGFBP1, POMC, APOA1, IL1RN, INS, LTA, MMP3, THBS2) were overlapped with data in Human Genome Epidemiology Network (HuGENet), in which they were identified as candidates for cardiovascular disease continuum.     

Long time evolution of atherosclerotic plaques

The evolution of atherosclerosis in general, and the influence of wall shear stress on the growth of atherosclerotic plaques in particular, is an intricate phenomenon which is still only partly understood. We therefore propose a qualitative mathematical model which consists of a number of ordinary differential equations for the concentrations of the most relevant constituents of the atherosclerotic plaque. These equations were studied both for the case that the wall shear stress is a parameter (model A), and for the case in which the plaque evolution is coupled to the blood flow (model B) which results in a time dependent wall shear stress. We find that both models exhibit a class of marginally stable equilibria, all reflecting states in which the plaque only grows for a short period of time after a perturbation. The uncoupled model A, however, shows bi-stability between this class of equilibria and another equilibrium state in which the plaque experiences unlimited growth in time, if the LDL cholesterol intake exceeds a threshold value. In model B the bi-stability vanishes, but we find that there is still a critical value of the LDL cholesterol intake beyond which the lumen radius drastically decreases. We show that this decrease is quite sensitive to the value of the wall shear stress.     

Assessment of atherosclerotic plaques in the rabbit abdominal aorta with interleukin-8 monoclonal antibody-targeted ultrasound microbubbles

In this study, we aimed to prepare a neovascularization-relevant inflammatory cytokine-targeted ultrasound contrast agent and apply it in the ultrasound imaging of atherosclerotic plaque. An interleukin-8 (IL-8) monoclonal antibody was conjugated to SonoVue microbubbles using the N-succinimidyl-3-(2-pyridyldithio)propionate cross-linking method. Then, a prepared IL-8-targeted contrast agent was used for contrast-enhanced ultrasound (CEU) to detect rabbit abdominal aorta atherosclerotic plaque and to investigate the imaging characteristics of atherosclerotic plaque with the contrast agent. We found that an IL-8 monoclonal antibody can be successfully coupled to SonoVue microbubbles with stable biological characteristics. CEU with this IL-8-targeted contrast agent can increase the atherosclerotic plaque detection sensitivity, with stronger echo, so that three more plaques were detected compared with using non-targeted SonoVue microbubbles. Thus, an inflammatory cytokine-targeting ultrasound contrast agent carrying IL-8 monoclonal antibody can provide unique advantages for researching the characteristics of atherosclerotic plaque.     

Fungal rDNA signatures in coronary atherosclerotic plaques

Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology.     

Initial stress in biomechanical models of atherosclerotic plaques

Rupture of atherosclerotic plaques is the underlying cause for the majority of acute strokes and myocardial infarctions. Rupture of the plaque occurs when the stress in the plaque exceeds the strength of the material locally. Biomechanical stress analyses are commonly based on pressurized geometries, in most cases measured by in-vivo MRI. The geometry is therefore not stress-free. The aim of this study is to identify the effect of neglecting the initial stress state on the plaque stress distribution. Fifty 2D histological sections (7 patients, 9 diseased coronary artery segments), perfusion fixed at 100 mmHg, were segmented and finite element models were created. The Backward Incremental method was applied to determine the initial stress state and the zero-pressure state. Peak plaque and cap stresses were compared with and without initial stress. The effect of initial stress on the peak stress was related to the minimum cap thickness, maximum necrotic core thickness, and necrotic core angle. When accounting for initial stress, the general relations between geometrical features and peak cap stress remain intact. However, on a patient-specific basis, accounting for initial stress has a different effect on the absolute cap stress for each plaque. Incorporating initial stress may therefore improve the accuracy of future stress based rupture risk analyses for atherosclerotic plaques.     

CD34 Class III positive cells are present in atherosclerotic plaques of the rabbit model of atherosclerosis

CD34 is a positive marker for haematopoietic stem cells and endothelial cells. Recent evidence suggests that haematopoietic progenitor cells are involved in atherogenesis. CD34-positive haematopoietic progenitor cells have never been described in rabbit atherosclerotic tissues. The aim of this study is to identify CD34-positive haematopoietic progenitor cells in rabbit atherosclerotic tissues, and to compare this with macrophage (RAM-11), alpha smooth muscle cell actin and fibroblast (prolyl-4-hydroxylase) immunoreactive cells. Sixteen Male New Zealand White rabbits were divided into two groups: Group 1, control diet (Con); group 2, 0.5% cholesterol diet, and killed after 12 weeks. Immunohistochemistry was used to detect CD34 haematopoietic progenitor cells. CD34-positive haematopoietic progenitor cells were identified both within and overlying atherosclerotic plaques. As well, these haematopoietic progenitor cells also stained for RAM-11, CD45, prolyl-4 hydroxylase and alpha smooth muscle cell actin. These findings suggest that in the rabbit model of atherosclerosis, the previously identified macrophages, smooth muscle cells and fibroblasts within and overlying atherosclerotic plaques might be of haematopoietic origin.     

Detection of nanobacteria-like particles in human atherosclerotic plaques

Recent and historical evidence is consistent with the view that atherosclerosis is an infectious disease or microbial toxicosis impacted by genetics and behavior. Because small bacterial-like particles, also known as nanobacteria have been detected in kidney stones, kidney and liver cyst fluids, and can form a calcium apatite coat we posited that this agent is present in calcified human atherosclerotic plaques. Carotid and aortic atherosclerotic plaques and blood samples collected at autopsy were examined for nanobacteria-like structures by light microscopy (hematoxylin-eosin and a calcium-specific von Kossa staining), immuno-gold labeling for transmission electron microscopy (TEM) for specific nanobacterial antigens, and propagation from homogenized, filtered specimens in culture medium. Nanobacterial antigens were identified in situ by immuno-TEM in 9 of 14 plaque specimens, but none of the normal carotid or aortic tissue (5 specimens). Nanobacteria-like particles were propagated from 26 of 42 sclerotic aorta and carotid samples and were confirmed by dot immunoblot, light microscopy and TEM. [3H]L-aspartic acid was incorporated into high molecular weight compounds of demineralized particles. PCR amplification of 16S rDNA sequences from the particles was unsuccessful by traditional protocols. Identification of nanobacteria-like particles at the lesion supports, but does not by itself prove the hypothesis that these agents contribute to the pathogenesis of atherosclerosis, especially vascular calcifications.     

Immunolocalization of A1 adenosine receptors in mammalian spermatozoa.

The presence of A1 adenosine receptors (A1AR) in mammalian spermatozoa was previously demonstrated by radiochemical and immunochemical detection. This study was performed to investigate the cellular location of the A1AR to determine whether these receptors were somehow connected with ecto-adenosine deaminase and to evaluate their function in calcium uptake. By immunofluorescence staining we showed that in mammalian spermatozoa A1AR were constantly localized in the acrosomal region. This finding was confirmed by immunogold detection. Confocal analyses with anti-A1 and anti-ADA antibodies showed a high degree of co-localization. Calcium loading assay showed that this association was functional and affected calcium accumulation in mammalian spermatozoa. Therefore, we concluded that the acrosomal localization of A1AR was a constant feature in mammalian sperm. Moreover, these A1 receptors were functionally coupled to ecto-ADA and were able to modulate calcium uptake into an IP3-gated store.(J Histochem Cytochem 48:1163-1171, 2000)     

Immunolocalization of fibroblast growth factor receptors 1 and 2 in mouse palate development

Recent evidence has implicated mutations of fibroblast growth factor receptors (FGF-R) in the pathogenesis of craniosynostotic syndromes. Cleft palate can be a component of such syndromes. The expression of FGF-R1 and FGF-R2 has been delineated in normally developing cranium, where they seem to regulate cellular differentiation and proliferation, respectively. The specific role of fibroblast growth factor signaling in mammalian palate development is unclear. The authors investigated the patterns of expression of FGF-R1 and FGF-R2 throughout mouse palatal development in the embryo. Time-dated CD-1 mouse heads (n = 135) were harvested at embryonic ages 12.5, 13.5, 14.5, 15.5, and 16.5 days (term gestation = 19.5 days), fixed in paraformaldehyde, embedded in paraffin, and sectioned. In addition, paired palatal shelves (n = 30) were isolated by means of microdissection from embryonic day--13.5 embryos, grown on Millipore filters in serum-free medium in vitro for 24, 48, 72, or 96 hours and processed for histological analysis. Immunohistochemical analysis for FGF-R1 and FGF-R2 was performed on the in vivo and in vitro specimens. FGF-R1 and FGF-R2 were found to be specifically expressed in the epithelium of the developing palatal shelves from the time of their outgrowth from the maxillary processes through completion of fusion in vivo and in vitro. Expression of both receptors was particularly strong during the phases of medial epithelial-medial epithelial contact between the individual shelves, through the formation of the medial epithelial seam, to the ultimate dissolution of the seam. Such a pattern of expression seems to implicate fibroblast growth factor signaling in the regulation of the critical phase of fusion of the bilateral shelves. The expression of both FGF-R1 and FGF-R2 in the lateral palatal mesenchyme, where such secondary structures as tooth primordia and bone begin to appear, also suggests a role for fibroblast growth factor signaling in the induction of ongoing differentiation and maturation of the palate after fusion. These data suggest that fibroblast growth factor signaling may play a role in the epithelial-mesenchymal interactions that dictate fusion and maturation of the developing palate. Furthermore, the data are consistent with the correlation of cleft palate formation with aberrant fibroblast growth factor signaling.     

Neutrophil infiltration and oxidant-production in human atherosclerotic carotid plaques

To clarify the clinical implications of neutrophils in vulnerable plaques we evaluated the function and activity of infiltrated neutrophils in an atherosclerotic plaque, focusing on oxidant production. A histopathological investigation was performed using carotid arterial samples obtained from seven patients. The atherosclerotic plaques were examined cytochemically for naphthol-ASD-chloroacetate esterase activity and oxidant-production, and immunohistochemically using N-formyl peptide receptor-like 1 (fPRL1)-, CD66b-, CD68- or p22phox-specific antibodies. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque was estimated using an activity index. Naphthol-ASD-chloroacetate esterase activity was found in cells located in the atherosclerotic plaque, indicating that the cells were neutrophils. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque decreased to approximately 60% of the intensity observed in the capillary vessels. Oxidant-production was also detected in the plaques, and both neutrophils and macrophages were observed at the corresponding oxidant-production sites. p22phox expression was also located in the same areas in which oxidant-production was observed in these plaques. We could not directly evaluate how much ROS generated from the infiltrated neutrophils contributed the plaque vulnerability followed by its rupture. However, the infiltrated neutrophils in the atherosclerotic plaques morphologically appeared activated and were actively generating oxidant, implying that neutrophils, together with macrophages, infiltrate into atherosclerotic plaques and contribute to plaque vulnerability.     

Differential beta-arrestin binding of AT1 and AT2 angiotensin receptors

Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, beta-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII-bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of beta-arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.     

Immunohistochemical detection of angiotensin receptors AT1 and AT2 in adrenal tumors

Angiotensin II is well known to affect the adrenal cell growth and function. Angiotensin receptors AT1 and AT2 were found to be present in the normal adrenal gland. However, the data on the expression of the angiotensin receptors in the adrenal tumors are very scarce. To overcome this gap, the paraffin sections of the adrenal cortical tumors and of pheochromocytomas from the archival material were immunostained with antibodies raised against AT1 (sc-1173) and AT2 (sc-9040) receptor proteins. In hyperplasia of the adrenal cortex and in benign adrenocortical adenomas, both functioning and non-functioning, the AT1 immunostaining was present mainly in the cell membranes. A positive immunoreaction was also found in the subpopulation of cell nuclei and within the cytoplasm. In the adrenal cancer, as well as in pheochromocytomas, neither cell membranes nor cell nuclei were immunostained with anti-AT1 antibody. However, a weak AT1 immunostaining was present within the cytoplasm of tumoral cells. With anti-AT2 antibody, in all tumors investigated, the tumoral cells were immunonegative but moderate to strong AT2 immunostaining was observed in the walls of intratumoral blood vessels and in the interstitial tissue. Our data indicates that the expression of AT1 receptors is altered in adrenal cancer and in pheochromocytomas. The expression of AT2 receptors, in turn, may be connected with the process of tumoral neo-angiogenesis.     

Immunolocalization of GnRHRI,gonadotropin receptors,PGR, and PGRMCI during follicular development in the rabbit ovary

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.     

Immunolocalization of full-length NK1 tachykinin receptors in human tumors.

Biological effects of substance P (SP) are mediated by the neurokinin-1 (NK1) receptor that exists as a full-length and as a carboxy-terminally truncated isoform in humans. Although NK1 receptor mRNA and binding sites have been detected in certain malignancies, little is known about the cellular and subcellular localization of NK1 receptor protein in human neoplastic tissues. We developed and characterized a novel anti-peptide antibody to the carboxy-terminal region of the human full-length NK1 receptor. Specificity of the antiserum was demonstrated by (1) detection of a broad band migrating at molecular mass 70,000-90,000 Da in Western blots of membranes from NK1-expressing tissues; (2) cell-surface staining of NK1-transfected cells; (3) translocation of NK1 receptor immunostaining after SP exposure; and (4) abolition of tissue immunostaining by preadsorption of the antibody with its immunizing peptide. Distribution of NK1 receptors was investigated in 72 formalin-fixed, paraffin-embedded human tumors showing that NK1 receptors were frequently expressed in glioblastomas and breast and pancreatic carcinomas. Immunoreactive NK1 receptors were clearly confined to the plasma membrane and uniformly present on nearly all tumor cells. Development of this novel NK1 receptor antibody allows the efficient localization of NK1 receptor protein in human formalin-fixed, paraffin-embedded tissues. NK1 receptor visualization with this simple and rapid immunohistochemical method will facilitate identification of tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention using SP analogs.     

ACE2 and AT4R are present in diseased human blood vessels

A growing body of evidence suggests that the angiotensin II fragments, Ang(1-7) and Ang(3-8), have a vasoactive role, however ACE2, the enzyme that produces Ang(1-7), or AT4R, the receptor that binds Ang (3-8), have yet been simultaneously localised in both normal and diseased human conduit blood vessels. We sought to determine the immunohistochemical distribution of ACE2 and the AT4R in human internal mammary and radial arteries from patients undergoing coronary artery bypass surgery. We found that ACE2 positive cells were abundant in both normal and diseased vessels, being present in neo-intima and in media. ACE2 positive immunoreactivity was not present in the endothelial layer of the conduit vessels, but was clearly evident in small newly formed angiogenic vessels as well as the vaso vasorum. Endothelial AT4R immunoreactivity were rarely observed in either normal and diseased arteries, but AT4R positive cells were observed adjacent to the internal elastic lamine in the internal mammary artery, in the neo-intima of radial arteries, as well as in the media of both internal mammary artery and radial artery. AT4R was abundant in vaso vasorum and within small angiogenic vessels. Both AT4R and ACE2 co-localised with smooth muscle cell alpha actin. This study identifies smooth muscle cell alpha actin positive ACE2 and AT4R in human blood vessels as well as in angiogenic vessels, indicating a possible role for these enzymes in pathological disease.