The association of matrix Gla protein isomers with calcification in capsules surrounding silicone breast implants

Implanted silicone medical prostheses induce a dynamic sequence of histologic events in adjacent tissue resulting in the formation of a fibrotic peri-prosthetic capsule. In some cases, capsular calcification occurs, requiring surgical intervention. In this study we investigated capsules from silicone gel-filled breast prostheses to test the hypothesis that this calcification might be regulated by the small vitamin K-dependent protein, matrix Gla protein (MGP), a potent inhibitor of arterial calcification, or by Fetuin-A, a hepatocyte-derived glycoprotein also implicated as a regulator of pathologic calcification. Immunolocalization studies of explanted capsular tissue, using conformation-specific antibodies, identified the mineralization-protective γ-carboxylated MGP isomer (cMGP) within cells of uncalcified capsules, whereas the non-functional undercarboxylated isomer (uMGP) was typically absent. Both were upregulated in calcific capsules and co-localized with mineral plaque and adjacent fibers. Synovial-like metaplasia was present in one uncalcified capsule in which MGP species were differentially localized within the pseudosynovium. Fetuin-A was localized to cells within uncalcified capsules and to mineral deposits within calcific capsules. The osteoinductive cytokine bone morphogenic protein-2 localized to collagen fibers in uncalcified capsules. These findings demonstrate that MGP, in its vitamin K-activated conformer, may represent a pharmacological target to sustain the health of the peri-prosthetic tissue which encapsulates silicone breast implants as well as other implanted silicone medical devices.     

Immunocytochemical investigation of atherosclerotic changes observed in aortocoronary bypass vein grafts using monoclonal antibodies

The authors have performed immunocytochemical surveys on atherosclerotic changes observed in saphenous vein aortocoronary bypass grafts, comparing the changes occurring in coronary and aortic lesions. The two monoclonal antibodies used in this study were obtained by T. Tsukada. One of them, named HHF35, exhibited specificity to smooth muscle cells; the other, named HAM56, was specific to macrophages. These immunocytochemical studies clearly demonstrated that cells encountered within the fibrous intimal thickening in the vein graft were inevitably smooth muscle cell in origin. Macrophages were seldom seen in the grafts examined. In contrast to vein grafts, macrophages were noted within the intima of all specimens from arterial atherosclerotic lesions obtained from the same patients. These studies suggest a difference in the progression of intimal thickening between the venous graft and the arterial atherosclerotic lesions.     

Development of complex atherosclerotic lesions in pigs with inherited hyper-LDL cholesterolemia bearing mutant alleles for apolipoprotein B.

The development of atherosclerotic lesions was studied in pigs aged 4 to 54 months with inherited hyperlow-density lipoprotein (LDL) and hypercholesterolemia (IHLC pigs). These pigs bear the Lpb5 and Lpu1 mutant alleles for apolipoproteins B and U and demonstrate spontaneously elevated cholesterol levels, due primarily to elevated LDL. By 1 year of age, IHLC pigs exhibited focal lesions in the major coronary, iliac, and femoral arteries that were composed of macrophage-derived from cells and smooth muscle cells. Peripheral arterial lesions were more fibrous than those found in the coronaries. By 2 years of age, complicated stenotic lesions containing fibrous caps, necrotic cores, cholesterol clefts, granular calcium deposits, and neovascularization deep within the lesion were common in the major coronary vessels. Peripheral vascular lesions were more smooth muscle cell-rich and fibrotic. By 3 years of age, neovascularization was observed throughout the intimal lesion, and hemorrhage and rupture were common. The extent of complicated lesion formation correlated with both the degree and duration of hypercholesterolemia, with the most stenotic lesions observed in the coronary arteries of the oldest animals having the highest cholesterol levels. Thus IHLC pigs with mutant apolipoproteins B and U develop complicated atherosclerotic plaques that closely resemble advanced atherosclerotic lesions found in humans.     

Expression of leptin receptor (Ob-R) in human atherosclerotic lesions: potential role in intimal neovascularization

Neovascularization of the adventitial vasa vasorum with extension into the intima of atherosclerotic lesions is frequently observed, but its pathophysiological significance is still subject to debate. Recently, leptin, the product of the Ob gene, was identified. Leptin, via activation of the endothelial receptor (Ob-R), generates a growth signal involving a tyrosine kinase-dependent intracellular pathway and promotes angiogenic processes. We hypothesized that a high concentration of leptin within vasa vasorum and plaque itself, may influence inflammatory and vascular neovascularization coupling with functional upregulation of the vascular endothelial growth factor (VEGF). Microscopic computerized tomography was utilized for the spatial distribution of vasa vasorum and intimal neovascularization from atherosclerotic human coronary arteries. Atherosclerotic coronary arteries showed a dense plexus of microvessels in the adventitia and plaque itself. Microscopic analysis from human atherosclerotic aortas revealed an increase in the intimal thickness with neovascularization. The immunoreactivity for Ob-R, VEGF and matrix metalloproteinase (MMP) increased in atherosclerotic plaque, predominantly in the endothelial lining of the intimal neovessel and macrophages/foam cells. Our observation of a prominent colocalization between Ob-R, VEGF and MMP supports this hypothesis and these factors participate in the neovascularization of atherosclerotic lesions. The present study is the first report on vascular tissue and it opens a promising perspective concerning future investigations of leptin-dependent modulation of atherogenesis and vascular neovascularization under pathophysiolgical conditions.     

Clonal mapping of the human aorta. Relationship of monoclonal characteristics, lesion thickness, and age in normal intima and atherosclerotic lesions.

The surfaces of 8 aortas from women heterozygous for G-6-PD isoenzymes were mapped for an examination of the relationships of monoclonality, lesion type, lesion thickness, and age of the patient. The percent B isoenzyme value of samples of normal intima (n = 315), fatty steak (n = 68), or fibrous plaque (n = 64) was used to define monoclonality, expressed as the [Z] score, the number of standard deviations from the mean percent B isoenzyme of samples of underlying media. Intimal thickness increased significantly with type of lesion, such that intima less than fatty streak less than fibrous plaque, and with the age of the patient. The percentage of monoclonal portions also increased with lesion type, such that 1% of samples of normal intima were monoclonal, compared with 4.4% of fatty streaks and 12.5% of fibrous plaques (P less than 0.005). Monoclonality increased with intimal thickness when normal intima, fatty streaks, and fibrous plaques were combined (P = 0.0001). When examined separately, normal intima showed a direct correlation between monoclonality and intimal thickness. In contrast, the monoclonality of fatty streaks was inversely associated with thickness (P = 0.016) and the monoclonality of fibrous plaques not related to thickness. When entered into a multiple regression model, lesion type and age, but not lesion thickness, significantly predicted monoclonality. The lack of association of intimal thickness with monoclonality suggests that it is the type of lesion that determines monoclonality and not merely its thickness. This implies that mechanisms other than clonal selection are responsible for the monoclonal characteristics of human atherosclerotic lesions.     

Nogo‐B expression,in arterial intima,is impeded in the early stages of atherosclerosis in humans

Nogo‐B (Reticulon 4B) is considered to be a novel vascular marker, which may have a protective role in injury‐induced neointima formation and atherosclerosis. Nogo A/B is found to be crucial for monocyte/macrophage recruitment in acute inflammation and it is expressed in CD68 + macrophages. We hypothesize that macrophage infiltration in atherosclerosis is not dependent on Nogo‐B expression in arterial wall. We have assessed Nogo‐B expression and macrophage accumulation in the iliac arteries of healthy organ donors and organ donors with cardiovascular risk factors. Paraffin sections of 66 iliac arteries, from 44 deceased organ donors (17 women and 27 men), were studied. The healthy and cardiovascular risk (CVR) subgroups were created. With regard to staging of the atherosclerotic process, the thickness of arterial intima was measured in digitalized images of H+E stained tissue sections. Immunohistochemical reactions (Nogo‐B and CD68) were carried out in all arteries (66 samples). Western blotting (WB‐19 samples) and real‐time PCR (27 samples) were performed on selected arteries. Significantly higher Nogo‐B expression was demonstrated in the intima of the healthy subjects' subgroup, using immunohistochemistry. WB and real‐time PCR revealed a trend toward lower Nogo‐B expression in the adventitia of the CVR subgroup. Furthermore, the thickness of the intima was found to negatively correlate with the expression of Nogo‐B in the intima and media (r = ?0.32; p < 0.05; r = ?0.32; p < 0.05). Macrophage infiltrates were more prominent in intima of CVR subjects (0.65 vs 3.52 a.u.; p < 0.01). Macrophage density in intima increased with atherosclerosis progression (r = 0.37; p < 0.01). CD68 macrophages density in adventitia was lower in CVR arteries than in healthy arteries. The expression of Nogo‐B, in arterial intima, is impeded in the early stages of atherosclerosis. Accumulation of arterial intimal CD68 macrophages has been shown to progress; however, the overall macrophage density in the adventitia is reduced in arteries shown to have intimal thickening. Macrophage infiltration is not accompanied by Nogo‐B expression in atherosclerotic arteries.     

Identification of platelet-derived growth factor A and B chains in human renal vascular rejection.

Platelet-derived growth factor (PDGF) exists as a dimer composed of two homologous but distinct peptides termed PDGF-A and -B chains, and may exist as AA, AB, and BB isoforms. The PDGF-B chain has been implicated as a mediator of renal vascular rejection by virtue of up-regulated expression of its receptor, PDGF beta-receptor, in affected arteries. A role for PDGF-A chain in mediating intimal proliferation has been suggested in human atherosclerosis (Rekhter MD, Gordon D: Does platelet-derived growth factor-A chain stimulate proliferation of arterial mesenchymal cells in human atherosclerotic plaques? Circ Res 1994, 75:410), but no studies of this molecule in human renal allograft injury have been reported to date. We used two polyclonal antisera to detect expression of PDGF-A chain and one monoclonal antibody to detect PDGF-B chain by immunohistochemistry in fixed, paraffin-embedded tissue from 1) normal adult kidneys, 2) a series of renal transplant biopsies chosen to emphasize features of vascular rejection, and 3) allograft nephrectomies. Immunohistochemistry was correlated with in situ hybridization on adjacent, formalin fixed tissue sections from nephrectomies utilizing riboprobes made from PDGF-A and -B chain cDNA. PDGF-A chain is widely expressed by medial smooth muscle cells of normal and rejecting renal arterial vessels of all sizes by immunohistochemistry and in situ hybridization. PDGF-A chain is also expressed by a population of smooth muscle cells (shown by double immunolabeling with an antibody to alpha-smooth muscle actin) comprising the intima in chronic vascular rejection. In arteries demonstrating acute rejection, up-regulated expression of PDGF-A chain by endothelial cells was detected by both immunohistochemistry and in situ hybridization. In contrast, PDGF-B chain was identified principally in infiltrating monocytes within the rejecting arteries, similar to its localization in infiltrating monocytes in human atherosclerosis. Although less prominent than the case for PDGF-A chain, PDGF-B chain also was present in medial and intimal smooth muscle cells in both rejecting and nonrejecting renal arteries. PDGF-A and -B chains have now been localized at both the mRNA and protein levels to the intimal proliferative lesions of vascular rejection. These peptides, which are known stimuli for smooth muscle cell migration and proliferation in experimental vascular injury, may have similar stimulatory effects on smooth muscle cells in an autocrine and/or paracrine manner to promote further intimal expansion and lesion progression in this form of human vasculopathy.     

Correlation of morphological variables in the coronary atherosclerotic plaque with clinical patterns of ischemic heart disease

The frequency and severity of "morphological" variables (fibrosis, proteoglycan accumulation, atheroma, intimal vascularization, calcification, acute intimal hemorrhage, and both adventitial and intimal lymphoplasmacellular infiltrates) in atherosclerotic plaques were related to plaque type, percentage of lumen reduction, plaque length, and intimal and medial thickness in 3,640 coronary artery sections sampled at the site of maximal lumen reduction in 8 selected segments from 100 cases of acute myocardial infarct, 50 of chronic angina, 208 of unexpected sudden coronary death with or without prodromata, and from 97 normal subjects dying accidentally. Morphological variables were occasionally observed in 1,519 sections with no lumen reduction. They were found only in sections from ischemic patients. With increasing luminal stenosis and intimal thickness, progression of the coronary plaque seemed to start as a fibrous change followed by proteoglycan accumulation in the deeper portion of the fibrous intima. Proteoglycan deposits appeared as a recurrent phenomenon. In them, atheroma or calcification develop. Intimal hemorrhage was a less frequent variable. It was found mainly in a vessel supplying an infarcted area. Lymphoplasmacellular inflammation correlated mainly with proteoglycan accumulation and atheroma, both showing a parallel increase with increasing intimal thickness and lumen reduction. No correlation was found between plaque variables and sex, age, heart weight, and infarct size. Significant variations in the distribution of plaque variables were observed among hearts of patients in the ischemic groups and between them and controls. In particular, inflammatory reaction was significantly more frequent and severe in ischemic groups than in controls, independent of the degree of coronary stenosis. Coagulative myocytolysis (contraction band necrosis), found in the majority of ischemic patients, correlated with the inflammatory reaction in supplying vessels. A peculiar tropism of mononuclear cell infiltrates for adventitial nerve structures was found. As a result, we question whether this inflammation may trigger coronary spasm and/or coagulative myocytolysis ("active" plaque vs "inactive" plaque).     

Expression of thrombospondins by endothelial cells. Injury is correlated with TSP-1.

The thrombospondins (TSP-1, -2, and -3) comprise a family of proteins that are homologous at the carboxy terminus but have unique sequences at the amino terminus that might be correlated with the regulation of cell behavior. To investigate the expression of TSP-1, -2, and -3 in endothelial cells, we examined developing murine blood vessels and human atherosclerotic plaques by in situ hybridization. The expression of TSP-1 was also characterized in cultured bovine aortic endothelial cells. Expression of TSP-2 was seen in the dorsal aorta as early as embryonic day 10; TSP-1 was not detected in endothelial cells until later stages, and TSP-3 was not apparent in the vasculature. In atherosclerotic specimens, TSP-1 mRNA was detected in many intraplaque microvessels and in the endothelium lining the atheromatous plaque; TSP-2 was absent from these regions. Cultured bovine aortic endothelial cells did not transcribe TSP-2 mRNA at detectable levels. There were high steady-state levels of TSP-1 mRNA in subconfluent bovine aortic endothelial cells before confluence and at the wound edge after injury of the cell monolayer, with maximal expression of TSP-1 in cultures at a time during which approximately 35% of the cells were in S phase. As the majority of these cells subsequently undergo mitosis, these data are consistent with TSP-1 as an inhibitor of endothelial cell proliferation that functions in G1. These results support the conclusion that, despite sequence homology, the TSPs have distinct functions in vascular biology.     

Apolipoprotein E localization in human coronary atherosclerotic plaques by in situ hybridization and immunohistochemistry and comparison with lipoprotein lipase.

Apolipoprotein E (apo E) mediates both lipid accumulation by and removal from cells and may be secreted by both macrophages and smooth muscle cells in vitro, but its cellular source in atherosclerotic plaques is not known. Lipoprotein lipase (LPL) also enhances cell lipid accumulation and is synthesized by macrophage foam cells in atherosclerotic plaques. To determine the cellular source of apo E in human coronary atherosclerotic lesions and its relationship to LPL synthesis, in situ hybridization and immunohistochemistry were performed on 12 atherosclerotic plaques and six nondiseased coronary artery segments from 10 cardiac transplant recipients. Apo E messenger RNA was localized to both non-foam cell and foam cell macrophages in plaques, but not to other cell types, and was not detected in nonatherosclerotic arteries. Half of the regions with non-foam cell macrophages expressed neither apo E nor LPL messenger RNA, whereas 86% of macrophage foam cell-containing regions contained both messenger RNAs. Polyclonal antisera raised against human apo E localized apo E protein to the surface of macrophages and surrounding matrix in plaques but not in control coronary segments. An LPL-specific monoclonal antibody demonstrated that, similar to apo E, LPL protein on foam cell and non-foam cell macrophages was detected in atherosclerotic lesions, but LPL was also localized to intimal muscle smooth muscle cells and was not distributed as widely in association with matrix as was apo E. The expression of both apo E and LPL in atherosclerotic lesions but not in normal intima suggest that these molecules play a role in lipid metabolism in atherosclerosis.     

Granzyme B in atherosclerosis and transplant vascular disease: association with cell death and atherosclerotic disease severity.

Apoptosis of intimal cells is an important contributor to the pathogenesis of atherosclerosis and transplant vascular disease (TVD). Since the activated immune response may be a key regulator of apoptosis in these lesions, we used immunohistochemistry to characterize the presence and localization of granzyme B, a major mediator of the cytotoxic immune response, in advanced atherosclerosis and TVD. Formalin-fixed, paraffin-embedded transverse sections from human left anterior descending coronary arteries were cut serially and stained with antibodies specific for granzyme B, smooth muscle alpha-actin, CD68, and CD3. The amount of granzyme B staining was semi-quantitated on a 0-5+/5+ scale. Also, TUNEL staining and in situ hybridization was performed to visualize cells undergoing cellular damage suggestive of apoptosis, and to localize granzyme B mRNA, respectively. Granzyme B localization was similar in both diseases. This protease was absent in arteries with mild atherosclerosis, but was abundant in the intima and media of vessels with advanced atherosclerosis and TVD. Within the intima, granzyme B localized to TUNEL-positive foam cells surrounding lipid-rich atheromas. Staining of serial sections with granzyme B and either smooth muscle alpha-actin, anti-CD68, or anti-CD3 showed that granzyme B localized to smooth muscle cells, macrophages, and T-cells. Further, in situ hybridization for granzyme B mRNA in TVD cases localized its expression to infiltrating leukocytes and not foam cells. In conclusion, the presence of granzyme B in advanced atherosclerotic lesions and TVD is associated with increasing disease severity and cell death. These observations suggest that granzyme B-mediated apoptosis may contribute to the pathogenesis of these diseases.     

Osteopontin and phospho‐SMAD2/3 are associated with calcification of vessels in D‐CAA,an hereditary cerebral amyloid angiopathy

In severe forms of cerebral amyloid angiopathy (CAA) pathology, vascular calcification has been observed in the cerebral cortex, both in vivo on MRI and CT, and post‐mortem using histopathology. However, the pathomechanisms leading to calcification of CAA‐laden arteries are unknown. Therefore, we investigated the correlation between calcification of cortical arterioles and several potential modulators of vascular calcification using immunohistochemistry in a unique collection of brain material of patients with a hereditary form of CAA, namely hereditary cerebral hemorrhage with amyloidosis‐Dutch type (HCHWA‐D or D‐CAA). We show a topographical association of osteopontin (OPN) and TGFβ signaling factor phospho‐SMAD2/3 (pSMAD2/3) in calcified CAA vessel walls. OPN and pSMAD2/3 gradually accumulate in vessels prior to calcification. Moreover, we found that the vascular accumulation of Collagen 1 (Col1), OPN and pSMAD2/3 immunomarkers correlated with the CAA severity. This was independently of the vessel size, including capillaries in the most severe cases. We propose that calcification of CAA vessels in the observed HCHWA‐D cases may be induced by extracellular OPN trapped in the fibrotic Col1 vessel wall, independently of the presence of vascular amyloid.     

Lipids in cells of atherosclerotic and uninvolved human aorta. III. Lipid distribution in intimal sublayers

The distribution, content, and composition of tissue and cellular lipids in intimal layers of unaffected and atherosclerotic human aorta were studied. Aortic tissue was divided into medial and intimal layers; the intimal layer was further separated into elastic-hyperplastic and musculo-elastic sublayers. Cells were isolated from both intimal layers by enzyme digestion. The lipids extracted from whole tissue and cells were separated by TLC and analyzed by scanning densitometry. The highest content of phospholipids (PhL), triglycerides (TG), cholesterol (C), and cholesteryl esters (CE) was detected in the elastic-hyperplastic layer of atherosclerotic plaque. However, taking into account that the elastic-hyperplastic layer of intima in lesioned areas was thickened, the lipid content per volume unit of both sublayers in fatty streaks and in plaques was equal. In the media underlying an atherosclerotic plaque, an increase in CE rather than in other lipid classes occurred. In the intima, an overall increase in PhL, TG, C, and CE content was found to display a constant ratio between these lipid classes, similar to that of low density lipoproteins (LDL). Cells isolated from atherosclerotic lesions had a higher lipid content than cells from areas of unaffected intima. However, the increase in the content of different lipid classes was not proportional, compared with tissue lipids. The content of PhL was the same, while an increase in TG, C, and CE was observed. The major contribution to excess cellular lipid accumulation in cells from atherosclerotic lesions was made by CE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atherosclerosis in Macaca mulatta: Histopathological,morphometric, and histochemical studies in aorta and coronary arteries of spontaneous and induced atherosclerosis

A comparative study of morphology, size, and histochemistry of the intimal lesions in aorta and coronary arteries of spontaneously occurring and cholesterol-induced atherosclerosis in rhesus monkeys has been carried out. A group of 30 normal monkeys was also investigated. Spontaneous atherosclerosis was noted in 10 of 55 adult monkeys autopsied serially; fatty streaks or atheroma in the aorta was noted in seven, fibrous plaque was noted in two, and diffuse intimal thickening was observed on one animal only. The coronary arteries showed fibrous intimal thickening without lipid in 8 of these 10 monkeys. There was fair to heavy deposition of acid mucopolysaccharides in the thickened intima along with proliferation of myointimal cells and collagen fibers. In the seven monkeys which were fed an atherogenic diet for 6 months, the aorta showed fatty streaking and atheroma in all animals. The coronary arteries also showed a variable degree of atherosclerosis but the lipid in the thickened intima was not marked. The atherosclerotic plaque height was not significantly greater than that in the spontaneous disease. These differences between spontaneously occurring and cholesterol-induced atherosclerosis in monkeys tend to indicate that the basic mechanism of lesion formation in the two states may be different.     

Expression of matrix Gla protein and osteonectin mRNA by human aortic smooth muscle cells.

BACKGROUND: Recent data indicate that matrix proteins such as matrix Gla protein (MGP) and osteonectin (ON) influence not only mineralization of vasculature but smooth muscle cell (SMC) differentiation. METHODS: We examined whether MGP and ON are expressed by human aortic SMCs in vivo using Northern blotting, in situ hybridization and immunohistochemistry. RESULTS: MGP and ON mRNAs were strongly expressed in the aorta without atherosclerosis from newborn and four young subjects up to 10 years old. In the aorta from 15 adult cases, MGP and ON mRNAs were decreased as atherosclerosis developed. We determined cell type and distribution of the MGP- and ON mRNA-expressing cells by in situ hybridization and immunohistochemistry. In the aorta obtained from newborn and young subjects, SMCs in the media and thin intima expressed MGP mRNA and, to a lesser extent, ON mRNA. In the adult aorta with fibrous thickening, MGP mRNA was expressed by intimal SMCs and subpopulation of medial SMCs. Osteonectin mRNA was expressed mainly by intimal SMCs and few medial SMCs. Double immunohistochemical staining revealed that both MGP- and ON protein-expressing cells were positive for anti-alpha-smooth muscle actin antibody, aortic SMCs. CONCLUSIONS: These results suggested that MGP and ON expression by aortic SMCs might be regulated by the degree of atherosclerosis and SMC differentiation in human aorta.     

Analysis of microsatellite instability and loss of heterozygosity in human aortic atherosclerotic lesions

The aim of the present study is to investigate whether microsatellite instability (MSI) and loss of heterozygosity (LOH) are involved in atherogenesis. Paraffin-embedded tissues of thoracic and abdominal aortas were collected from 29 non-neoplastic autopsied cases. We selected two types of atherosclerotic lesions including fibrocellular intimal thickening and uncomplicated atheromatous lesions, and analyzed two sites such as the aortic tunica intima and tunica media in each case. The frequencies of MSI and LOH were highest in the aortic tunica intima of atheromatous lesions and lowest in the aortic tunica media of fibrocellular intimal thickening lesions. Our results suggest that genetic instabilities such as MSI and LOH may be involved in the development of atherosclerotic lesions.     

Immunology of atherosclerosis. Demonstration of heat shock protein 60 expression and T lymphocytes bearing alpha/beta or gamma/delta receptor in human atherosclerotic lesions.

Our previous work revealed the presence of a great number of activated T lymphocytes in early human atherosclerotic lesions, and we were able to induce atherosclerosis in normocholesterolemic rabbits by immunization with Mycobacterium tuberculosis heat-shock protein (HSP) 65. We hypothesized this latter phenomenon to arise from cross-reactivity of mycobacterial HSP 65 with the endogenously expressed homologous 60-kd form of this stress protein. To study HSP 60 expression and the phenotype of intima infiltrating T lymphocytes relative to the T cell receptor (TCR) in human atherosclerotic lesions, specimens of aorta, carotid arteries, and internal mammary arteries and veins, as well as saphenous veins and vena cava from 27 subjects, aged 23 to 80 years, were examined using immunohistochemical and immunofluorescence techniques on serial frozen tissue sections. HSP 60 was detected on endothelium, smooth muscle cells, and/or mononuclear cells of all carotid and aortic specimens, whereas vessels of smaller diameter, serving as reference specimens for normal intima without atherosclerotic lesions and mononuclear infiltration, showed no detectable expression of this stress protein. Furthermore, although the majority of CD3+ cells within the mononuclear cell infiltrates of atherosclerotic lesions bear the alpha/beta TCR, a considerable portion also consisted of gamma/delta TCR+ cells. Thus, 9.7% of T cells in the transition zone between normal intima and fatty streaks carry the gamma/delta TCR, a proportion that decreases to 6.6% and 4.3% in fatty streaks and atherosclerotic plaques, respectively. We conclude that the intensity of HSP 60 expression correlates positively with the atherosclerotic severity and that most lymphocytes participating in atherogenesis bear the alpha/beta TCR, although gamma/delta TCR+ cells are also enriched in atherosclerotic lesions. Expression of HSP 60 by intimal cells, caused, eg, by hemodynamic shear forces, may be responsible for recruitment of HSP-sensitized T cells, thus leading to the induction of an initiating inflammatory process in atherosclerosis. Other risk factors, such as high serum cholesterol levels, contribute to the final outcome of the disease.     

Activation of adventitial fibroblasts contributes to the early development of atherosclerosis: a novel hypothesis that complements the "Response-to-Injury Hypothesis" and the "Inflammation Hypothesis"

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This article introduces the hypothesis that the activation of the adventitia, specifically the fibroblasts, contributes to the formation of intimal atherosclerotic lesions. The evidence for this hypothesis includes: (a) the early proliferative changes seen in fibroblasts found in the adventitia; (b) the increase and the alteration of extracellular matrix deposition in the adventitia; (c) fibroblast differentiation into myofibroblasts and migration into the intima; and (d) fibroblast synthesis and release of cytokines that have potent effects on neighboring smooth muscle and endothelial cells prior to intimal lesion formation. In conclusion, the activation of adventitial fibroblasts is a key regulator of vascular function and structure from the "outside-in" and contributes to the development of atherosclerotic lesions. The outer location of the adventitia makes it a suitable location for drug delivery and gene therapy aimed at preventing and treating atherosclerosis.     

Combination of sodium thiosulphate, cinacalcet, and paricalcitol in the treatment of calciphylaxis with hyperparathyroidism

Calciphylaxis (calcific uremic arteriolopathy) is a severe complication of hemodialysis characterized by subcutaneous calcification of the small arteries and tissue necrosis. Our case report is focused on a woman receiving hemodialysis (HD) with diabetes mellitus for 20 years and severe secondary hyperparathyroidism, who presented painful subcutaneous nodules, skin necrosis and ulcerations. As the treatment of calciphylaxis is mainly empirical and controversial, we decided to administer cinacalcet with paricalcitol for the control of hyperparathyroidism and sodium thiosulfate to improve the calcification of the arterioles. Two months after the start of the therapy, parathyroid hormone (PTH) decreased significantly and the skin lesions nearly disappeared. Thus, we believe that the combination of sodium thiosulfate with cinacalcet and paracalcitol is effective for the treatment of calciphylaxis with secondary hyperparathyroidism.     

Progesterone receptors in the human heart and great vessels

Progesterone receptors (PgR) were identified in 31 of 50 specimens of human (men and women) thoracic ascending aorta, internal carotid, coronary artery, and left atrial appendage. This was accomplished with a peroxidase-antiperoxidase immunocytochemical assay employing a highly specific monoclonal antibody to primate PgR. In the aorta, specific staining was seen in the nuclei of smooth muscle cells and endothelium of intima, media, and adventitia. In the myocardium, staining was localized to the nuclei of the myocardial fibers. In internal carotid and coronary arteries, PgR was localized to endothelial nuclei of intima, and in vascular channels within the atherosclerotic plaques. PgR was also visible in the smooth muscle cell nuclei of uninvolved media and intima and at the plaque periphery. In contrast, receptor was not identified in vessels of the human uterus, breast, prostate, kidney, or gastrointestinal tract. These findings suggest that the heart and great vessels are target organs for steroid hormones.