Localization of collagen types I, III, IV and V, fibronectin and laminin in human arteries by the indirect immunofluorescence method

The distribution of types I, III, IV and V collagen and of the glycoproteins fibronectin and laminin in sections of human aortas, arteries and atherosclerotic plaques were studied using monospecific antibodies and indirect fluorescence microscopy. Types IV and V collagen and laminin were present in a narrow zone, representing the basement membrane, apposed to the endothelial layers of all these tissues. Types I and III collagen and fibronectin were located in the interstitial spaces of the intima and the media of blood vessels walls, whereas types IV and V collagen and laminin were found in the basement membranes underlying smooth muscle cells in these areas. Two types of atherosclerotic plaques were observed. Lipid-rich plaques contained less collagen and reduced amounts of the glycoproteins. Fibrous plaques consisted of regions deficient in types I and III collagen and collagen-rich regions with elevated levels of these two collagens as well as more fibronectin. The collagen-rich regions of fibrous plaques contained, however, little type IV and type V collagen and little of the glycoproteins laminin and fibronectin. This may be due to the reduced number of cells involved in the biosynthesis of these basement membrane proteins.     

Alterations of endothelin-converting enzyme expression in early and advanced stages of human coronary atherosclerosis

Endothelin-1 is a potent vasoconstrictor and exhibits a mitogenic activity on vascular smooth muscle cells (SMCs). Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin-1 processing. We studied the immunolocalization of ECE in human coronary atherosclerotic lesions with different disease stages. Frozen sections of normal coronary arteries with diffuse intimal thickening (n=13) and those of coronary arteries with early (n=10) or advanced atherosclerotic plaques (n=13) were studied. Monoclonal antibodies used were directed against SMCs, macrophages, endothelial cells, and ECE. For the identification of cell types that express ECE, double immunostaining analysis was also used. In normal coronary arteries, ECE immunoreactivity was observed in luminal endothelial cells and medial SMCs. Early atherosclerotic plaques, which consisted predominantly of SMCs, showed enhanced ECE expression in luminal endothelial cells and intimal SMCs. In advanced atherosclerotic plaques, distinct ECE expression was found in accumulated macrophages and in endothelial cells of intraplaque microvessels, while luminal endothelial cells showed relatively weak immunoreactivity for ECE. In conclusion, the present study demonstrates that the major cell types expressing ECE within the plaques are different between early and advanced stages of human coronary atherosclerosis. Enhanced ECE expression and possible endothelin-1 generation may contribute to SMC proliferation and vasoconstriction in early atherosclerotic stages, and may promote plaque destabilization in advanced atherosclerotic stages.     

Immunohistochemical localization of pepsinogen A and C containing cells in Barrett's oesophagus

Summary No data are available on the localization of Pepsinogen A (PGA=PG I) and Pepsinogen C (PGC=PG II) positive cells in Barrett's epithelium. Endoscopic biopsy specimens were taken from the columnar epithelium from 23 patients (n=93), and in addition from the cardia from eight healthy control subjects (n=38). The tissue was stained by the immunoperoxidase technique with specific anti-pepsinogen antisera, and double immunostained for PGA and PGC. In the Barrett's epithelium PGA was found in 28 out of 93 biopsy specimens (30.1%) and PGC in 55 out of 93 (59.1%). Chief cells always stained both for PGA and PGC, while clear mucous cells were often PGA– and PGC+. PGA+ and PGC+ cells were found each in 100% of the biopsy specimens with fundic type epithelium, in 21.7% and 70.7% of biopsy specimens with junctional type, in 0% and 26.1% of biopsy specimens with specialized epithelium and in 12.5% and 43.5% of biopsy specimens with mixed junctional/specialized features respectively. Dysplastic epithelium stained always negatively with both anti-pepsinogen antisera. In most control cardia biopsy specimens PGA as well as PGC were demonstrable; occasionally clear mucous glands were PGA– and PGC+.It is concluded that pepsinogen-containing cells can be accurately identified in the Barrett's epithelium; their presence seems related to the histological cell type. Identification of pepsinogen positive cells may contribute to a more accurate morphological classification of the Barrett's epithelium.Presented in part at the Annual Meeting of the American Gastroenterological Association, San Francisco, May 1986     

Quantification of cell types throughout the cycle of the human seminiferous epithelium and their DNA content

Summary The numbers of each different cell type in the human seminiferous epithelium were determined throughout the 6 stages of the cycle in both semithin and ultrathin sections obtained from 15 young adult men with normal testicular histology. Up to 4 types of A spermatogonia (Ad, Ap, Al and Ac) were distinguished. In addition, the DNA nuclear content of seminiferous epithelium cells was determined on Feulgen-stained sections. Both Ad and Ap spermatogonia showed a 2c DNA content and were present in the 6 stages of the cycle, though their numbers decreased in stages III–V. Both Al and Ac spermatogonia showed a DNA content varying from 2c to 4c. Al spermatogonia were observed in stages III–V; their numbers plus those of Ad spermatogonia in these stages were similar to the numbers of Ad spermatogonia in the other stages lacking in Al spermatogonia. Ac spermatogonia appeared in stages III–VI and their numbers plus those of Ap spermatogonia in stages III–V were similar to the numbers of Ap spermatogonia in the other stages lacking in Ac spermatogonia. The results suggest that Ad spermatogonia are the stem cells. Some of them replicate their DNA; during this replication they appeared as Al spermatogonia. Al spermatogonia divide, giving rise to both Ad and Ap spermatogonia. Some Ap spermatogonia replicate their DNA; during this process they are transformed into Ac spermatogonia which divide, giving rise to B spermatogonia.This work was supported by a grant from the Comisión Asesora de Investigación Científica y Técnica Madrid, Spain     

Transdifferentiation of smooth muscle cells into chondrocytes in atherosclerotic arteries in situ: implications for diffuse intimal calcification

Several hypotheses have been offered to explain the occurrence of arteriosclerotic calcification but the mechanisms involved are still not well understood. Using a combination of electron microscopy and immunohistochemistry, atherosclerotic plaques from human arteries as well as atherosclerotic-like lesions from aortas of apo-E-deficient mice were examined to identify cell type(s) associated with calcification. Electron microscopic analysis showed that, in human atherosclerotic plaques, chondrocyte-like cells were present in areas surrounding the necrotic cores. In these areas, some smooth muscle cells displayed features of their transdifferentiation into chondrocyte-like cells. Immunohistochemical analysis confirmed that smooth muscle cells with a reduced content of alpha-smooth muscle actin expressed Sox-9. Destruction of chondrocytes resulted in the accumulation of numerous membrane-bound vesicles in the extracellular space. Membrane-bound vesicles originating from chondrocytes were found to undergo calcification. Similar processes were found to occur in atherosclerotic-like lesions in apo-E-deficient mice. These observations suggest that transdifferentiation of smooth muscle cells into chondrocytes contributes to atherosclerotic calcification.     

Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy.

Extracellular matrix is the principal component of the fibrous caps of atherosclerotic plaques and intimal hyperplastic lesions of reconstructed arteries. Interstitial collagen form an important part of the matrix, and the balance between collagen synthesis and degradation by interstitial collagenase (matrix metalloproteinase-1, MMP-1) may determine whether plaques rupture or vessels develop stenosis. We examined type I procollagen gene expression in human atherosclerotic and restenotic carotid arteries using in situ messenger RNA (mRNA) hybridization and the expression of MMP-1 and its endogenous inhibitor (tissue inhibitor of metalloproteinases-1, TIMP-1) by immunohistochemistry. Compared with normal arteries, atherosclerotic plaques bed increased expression of immunoreactive MMP-1 and TIMP-1 with modest increase of type 1 procollagen mRNA. Early restenotic lesions (< 1.5 years) contained abundant type I procollagen mRNA but little immunoreactive MMP-1 and TIMP-1. Late restenotic lesions (> 4 years) resembled atheroma and exhibited increased immunoreactive MMP-1 and TIMP-1 as well as abundant type I procollagen mRNA. Compared with atherosclerotic plaques, type I procollagen is increased and MMP-1 is decreased in early restenotic lesions. MMP-1 and TIMP-1 expressions are upregulated in lesions with a clear atheroma. These findings suggest that the balance between proteolysis and matrix synthesis may influence both the stability of atheromatous plaques and the development of restenotic lesions.     

MAST CELL DISTRIBUTION,ACTIVATION, AND PHENOTYPE IN ATHEROSCLEROTIC LESIONS OF HUMAN CAROTID ARTERIES

Immunohistochemical staining for mast cell tryptase and chymase was used to examine the distribution, activation, and tryptase/chymase phenotype of mast cells (MCs) in 250 samples of atherosclerotic lesions (type I to VI) of human carotid arteries. Dual immunolocalization and histochemical techniques were used to identify the associations of MCs with macrophages, smooth muscle cells, and extracellular matrix components. Whereas normal carotid arteries contained very few MCs within the intima, atherosclerotic lesions showed increased MC numbers with variable focal accumulations. MCs were identifiable from the earliest stages of atherosclerosis, and especially at the shoulder regions of the fully formed atheroma. They were observed in close association with macrophages (HAM56 positive) and extracellular lipid, as well as at sites of foam cell formation. MCs and diffuse tryptase staining were also evident within sites of new calcification and around small calcified deposits. Extensive MC activation/degranulation, as judged by diffuse extracellular tryptase staining, was a common feature of the advanced atherosclerotic plaques complicated by fissure, haemorrhage, and thrombus formation. Moreover, such sites of extracellular MC tryptase were often associated with localized oedema and disruption of the stromal matrix. MCs which contained both tryptase and chymase (the MC_TC phenotype) represented approximately 80–95 per cent of all MCs. These studies are the first to demonstrate significant numbers and focal accumulations of MCs in all developmental stages of atherosclerotic carotid arteries. Since MCs contain or express a variety of potent mediators, their release could profoundly influence the development and pathological complications of atherosclerotic plaques. © 1997 John Wiley & Sons, Ltd.     

C-reactive protein in vulnerable coronary plaques

BACKGROUND: An increased level of serum C-reactive protein (CRP) is a known prognostic factor for acute coronary events and sudden cardiac death, and it is associated with coronary calcification. CRP is expressed in coronary arteries, but its role in the development of coronary plaques is unclear. AIM: To investigate CRP immunoreactivity in relation to the severity of coronary artery disease and plaque morphology in human left anterior descending coronary arteries (LAD). METHODS: A prospective, consecutive autopsy series of 66 patients (mean age 63.4 years) in Tampere University Hospital, Tampere, Finland. RESULTS: CRP immunoreactivity was seen in 59% of the cases. In logistic regression analysis with age, sex and body mass index as confounders, CRP immunoreactivity in LAD was associated with >50% stenosis and plaque calcification. All three cases with acute coronary thrombosis due to rupture or erosion of the plaque showed a clear immunopositive reaction. CRP-positive cells were never detected in normal arteries, but were often found in early fibrous plaques (75%) and almost invariably present in the shoulder area of plaques with necrotic core (96%). CRP immunoreactivity adjacent to calcified areas in more stable plaques (71%) was less consistent with one-third of these plaques showing no immunoreactivity. CONCLUSIONS: CRP immunoreactivity is associated with the progression of atherosclerosis, and especially with unstable coronary plaques. The immunoreactivity could cease at the stable calcified stages of atherosclerosis.     

Atherosclerosis of the carotid arteries in young patients with familial hypercholesterolemia

Summary Patients with heterozygous and homozygous Familial Hypercholesterolemia exhibit a high incidence of premature coronary heart disease, presumably due to atheromatous plaque-formation in the coronary arteries. Clinical symptoms develop when the disease has progressed to more severe stages of atherosclerosis. Aim of our study was to visualize and document early atheromatous lesions in the carotid arteries of asymptomatic patients with familial hypercholesterolemia under 30 years of age by Duplex-scan.Of 44 patients, 70% had detectable carotid plaques, while only 12% of the controls were affected. All patients with severe carotid disease had serum cholesterol levels above 350 mg/dl. In the age group 2–20 years, 66% of the patients exhibited plaques. Only 6% of the FH patients 21–30 years had normal carotid arteries. We conclude, that the process of atheromatous plaque formation in patients with FH starts early in life, severity of atherosclerosis being a function of both extent and duration of hypercholesterolemia. Duplex-scan examination of the carotid arteries is an efficient and precise non-invasive method suitable to visualize this process and, by measuring regression, monitor the efficacy of therapeutic measures.Abbreviations FHC Familial Hypercholesterolemia - LDL Low Density Lipoprotein - CHD Coronary Heart Disease This work was supported by a grant from Deutsche Forschungsgemeinschaft     

Monoclonality of smooth muscle cells in human atherosclerosis.

Atherosclerotic plaques contain a large monoclonal population of cells. Monoclonality could arise by somatic mutation, selection of a pre-existing lineage, or expansion of a pre-existing (developmental) clone. To determine the monoclonal cell type in plaque and learn when monoclonality arises, we studied X chromosome inactivation patterns using methylation of the X-linked human androgen receptor gene. Assays based on polymerase chain reaction were performed on samples of known cellular composition, microdissected from histological sections of human arteries. In atherosclerotic vessels, the majority of medial samples (7/11 coronary and 2/3 aortic) showed balanced (paternal and maternal) patterns of X inactivation, indicating polyclonality. In contrast, most samples of plaque smooth muscle cells showed a single pattern of X inactivation (3/4 aortic plaques and 9/11 coronary plaques; P < 0.01 versus media), indicating that plaque smooth muscle cells are monoclonal. Samples of plaque containing inflammatory or endothelial cells showed balanced X inactivation, also demonstrating polyclonality. Multiple plaques from a given patient showed no bias toward one allele, indicating there was no X-linked selection of cells during plaque growth. To determine whether plaques might arise from pre-existing clones (large X inactivation patches), we then studied 10 normal coronaries with diffuse intimal thickening. Six of the ten coronaries showed skewed X inactivation patterns in normal media and intima, suggesting the patch size in normal arteries is surprisingly large. Thus, smooth muscle cells constitute the monoclonal population in atherosclerotic plaques. The finding that normal arteries may have large X inactivation patches raises the possibility that plaque monoclonality may arise by expanding a pre-existing clone of cells rather than generating a new clone by mutation or selection.     

Functional heterogeneity of human antigen-presenting cells: Presentation of soluble antigen but not self-Ia by monocytes

These studies were undertaken to examine the phenotype of the antigen-presenting cells (APC) circulating in human peripheral blood. Cells adherent to glass were found to be efficient APC, restoring antigen-induced³H-thymidine incorporation to T4-positive T cells that had been rigorously depleted of contaminating APC. In order to identify the APC within the glass-adherent cells (AC), these cells were stained with a number of monocyte-specific monoclonal antibodies (Mo-Mab) including 3C10, 63D3, and 61D3, and the Mo-Mab-positive and -negative cells were separated with the fluorescence-activated cell sorter. This method of preparation yielded Mo-Mab(+) AC populations that were more than 98% positive for the relevant Mab when reanalyzed with the fluorescence-activated cell sorter. Less than 1% of the Mo-Mab(–) AC populations were positive when reanalyzed with the Mab used for the separation. However, each Mo-Mab(–) AC population was contaminated with variable numbers (4–60%) of Mo as detected by morphologic criteria, histochemical analysis for esterase activity, or staining with a different Mo-Mab. Both Mo-Mab(+) and (–) AC populations were found to be similarly effective APC, with as few as 500 cells/well supporting responses to streptokinase-streptodornase, tetanus toxoid, andCandida albicans antigen. In the absence of antigen, only 3C10(–), 63D3(–), or 61D3(–) AC consistently stimulated³H-thymidine incorporation of autologous T4 cells; large numbers (>5×10³/well) of APC were necessary to induce this response. These results support the conclusion that cells identified by Mo-specific Mab are capable of functioning as APC, inducing³H-thymidine incorporation in response to exogenous antigens. However, Mo-Mab(+) AC are not unique in this activity since Mo-Mab(–) AC also appeared to be able to present antigen. These Mo-Mab(–) AC appear to contain the majority of cells inducing autologous T4-cell reactivity.     

Cation flux studies of the lesion induced in human erythrocyte membranes by the thermostable direct hemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, an important agent of seafood-borne gastroenteritis, expresses several putative virulence factors that could account for the disease symptoms of infected humans, namely, diarrhea, nausea, and abdominal cramps. The pathogenicity of V. parahaemolyticus correlates well with the Kanagawa phenomenon (the hemolytic ability of strains grown on Wagatsuma blood agar), implicating the thermostable direct hemolysin (TDH) as the predominant toxin responsible for pathogenicity. TDH-induced hemolysis could be inhibited by the addition of the osmolyte sorbitol to the extracellular solution, supporting the hypothesis that hemolysis occurs through colloid osmosis secondary to an increase in the cation permeability of the membrane. The effect of TDH on cation permeability was investigated by measuring K+ (congener, 86Rb+) influx into human erythrocytes in which the endogenous cation transporters had been blocked (by use of ouabain, bumetanide, and nitrendipine). TDH increased K+ influx into these cells; this increase was rapid in onset and constant in magnitude, suggesting a direct action by TDH on the membrane. The kinetics of leak generation were examined; the relationship between counts accumulated and hematocrit indicated that the TDH-induced lesion is multihit in nature. TDH-induced K+ influx was sensitive to Zn2+. Time courses of hemolysis in isosmotic solutions of monovalent cation chlorides were used to obtain the selectivity series for the TDH-induced leak: Cs+ > Li+ > K+ > Rb+ > Na+. Both the Zn2+ sensitivity and this selectivity series were obtained for crude culture supernatants, suggesting that TDH is the predominant leak-inducing agent. Thus, we have identified several features of the TDH-induced leak likely to be important in the diarrhetic action of V. parahaemolyticus in the human intestine.     

T-cell receptor heterogeneity of gamma delta T-cell clones from human female reproductive tissues.

gamma delta T cells were isolated from human decidua parietalis, decidua basalis and cervix and cloned in the presence of interleukin-2 (IL-2). T-cell receptor (TcR) expression was then analysed and compared with that of a panel of gamma delta T-cell clones from peripheral blood. Only 17/40 (42.5%) clones from decidua parietalis were V gamma 9+/V delta 2+ as compared to 68/94 (72%) of peripheral blood clones (P < 0.005). Conversely, 50% of clones from decidua parietalis but only 15% of clones from peripheral blood were V delta 1+ (P < 0.001). At least seven distinct TcR types were identified among the panel of clones from decidua parietalis and at least six different types were expressed by the panel of 17 clones from cervix. This receptor heterogeneity was not a result of interdonor variation as in all instances where more than one clone was obtained from a single sample, individual clones having between two and five receptor types were identified. However, 23/24 (95.8%) of clones from decidua basalis were V gamma 9+/V delta 2+. Most clones from decidua parietalis and cervix, whether V gamma 9+/V delta 2+ or V delta 1+, were positive for the mucosal lymphocyte marker, HML-1, but expression was often heterogeneous within a single clone. In contrast, almost all gamma delta T-cell clones from peripheral blood were HML-1-. Thus, unlike the mouse, gamma delta T cells within these human female reproductive tissues have a diverse TcR repertoire which, in decidua parietalis, is distinct from that of peripheral blood.     

The properties of the inward rectifier potassium currents in rabbit coronary arterial smooth muscle cells

In freshly-isolated, single, smooth muscle cells of rabbit coronary arteries, an inward rectifier K⁺ current [I _K(IR)] was identified using the whole-cell voltage-clamp technique. The current/voltage (I/V) relationship of I _K(IR) showed strong inward rectification with a very small outward current when the smooth muscle cells were dialyzed with a pipette solution containing Mg²⁺. However, dialyzing the cells with a nominally Mg²⁺-free pipette solution revealed a significant outward current hump in the I/V relation of I _K(IR), suggesting that the strong inward rectification of I _K(IR) is partly due to the inhibitory effects of internal Mg²⁺. I _K(IR) was unaffected by tetraethylammonium (1 mM), 4-aminopyridine (1 mM), or glibenclamide (1 μM), but was inhibited by extracellular Ba²⁺ with a concentration of 0.87 μM eliciting half-maximal inhibition at –120 mV. I _K(IR) induced in rabbit coronary smooth muscle cells declined during very negative hyperpolarizing steps, due largely to a block by external Na⁺. I _K(IR) was inhibited by α₁-adrenergic stimuli. Methoxamine, an α₁-adrenergic agonist, concentration dependently inhibited I _K(IR) in the presence of the β-adrenergic antagonist propranolol. The methoxamine concentration required for half-maximal inhibition was 205 μM. We conclude that inward rectifier K⁺ current is present in rabbit coronary smooth muscle cells and that it shares many properties with the inward rectifier K⁺ current described for other cell types. Received: 2 February 1999 / Accepted: 24 March 1999     

Evidence for neoplastic cell differentiation in mediastinal T lymphoblastic lymphoma

Summary Immunostaining of frozen sections from a mediastinal T lymphoblastic lymphoma (T-LL) revealed the existence of two neoplatic cell populations characterized by different degrees of maturation. Several large nodules of 3A1 +/T11 +/T9 + T6–/T4–/T8–/T3– lymphoid cells, resembling normal early thymocytes, were surrounded by 3A1+/T11+/T9+/T6+/T4+/T8+/T3– cells resembling normal cortical thymocytes. The junctional area between early and cortical lymphocytes was occupied by numerous Leu-M3 +/PAM-1 + / DR + reticular macrophages which were also characterized by J5 reactivity. Cytokeratin +/keratin + epithelial cells were absent. Immunostaining of paraffin sections and of cytocentrifuge smears obtained from tumour cell suspensions revealed that a consistent percentage (8%) of neoplastic lymphoblasts were S-100+. Our findings are consistent with a cortical T-LL presenting areas of dedifferentiated cells or, alternatively, with an early T-LL whose cells were able to differentiate into cortical thymocytes, perhaps through the interaction with a specialized subset of J5 + macrophages.Supported by CNR contract N. 85.02020.44 Progetto Finalizzato Oncologia     

Immunohistochemical localization of heat shock protein-70 in normal-appearing and atherosclerotic specimens of human arteries.

Heat shock proteins (HSPs) are synthesized by cells under metabolic stress and are known to enhance a cell's ability to survive life-threatening stress. The authors have begun to examine HSPs in the context of human atherosclerosis. This study demonstrated immunohistochemically the presence of HSP-70 in human and rabbit arteries, and its distribution in relation to necrosis and lipid accumulation, as well as vascular smooth muscle cells and macrophages, in human atherosclerotic plaques. Advanced lesions from 10 human carotid endarterectomy specimens were compared with 11 human aortic specimens from autopsy and 8 rabbit aortas. The immunostaining procedure used a mouse monoclonal antibody specific for the inducible form of HSP-70. Normal rabbit aortas were tested for changes in HSP-70 up to 24 hours after removal, and were used as controls for the human aortas. Representative plaques were examined for lipid content by osmium staining, and for smooth muscle cell and macrophage components using cell-specific monoclonal antibodies followed by immunostaining. The results indicated that HSP-70 was present in human and rabbit arteries and remained unchanged in distribution or concentration up to 15 hours after death. HSP-70 was present weakly throughout the media of normal-appearing arterial specimens. In contrast, HSP-70 was concentrated in the central portions of more thickened atheromas around sites of necrosis and lipid accumulation. Macrophages were coincident with these areas and were observed to be lipid-loaded. In contrast, patches of smooth muscle cells were observed in very complicated plaques, but without consistent association with necrosis or increased HSP-70; plaque smooth muscle cells also were observed to contain lipid. Large, relatively avascular and collagenous areas of plaque also were occasionally positive for HSP-70 staining. The results support the hypothesis that elevated HSPs indicate which plaque cells, particularly macrophages, are more stressed in the depth of atheroma, especially in association with necrosis, and should prompt further investigation of the significance of HSP accumulation to the evolution of atherosclerotic plaques.     

Cytoskeletal features of normal and atheromatous human arterial smooth muscle cells

The distribution of actin, vimentin, desmin, and tropomyosin was studied in the media of the human aorta and femoral and coronary arteries, as well as in atheromatous plaques from the same arteries, by means of immunofluorescence, densitometric analysis of sodium dodecylsulfate-polyacrylamide gel electrophoresis, and bidimensional gel electrophoresis. The proportions of desmin-containing cells varied in the media of different arteries; 4 per cent of the cells in the aorta, 11 per cent in the coronary artery, and 37 per cent in the femoral artery contained desmin. In fibrous atheromatous plaques, independently of the artery, desmin-containing cells were almost absent, but they reappeared in complicated lesions. The content of vimentin per smooth muscle cell increased in fibrous atheromatous plaques, whereas the content of actin and tropomyosin was less than in normal media. Moreover, the alpha-actin predominance observed in the media was transformed to beta-actin predominance in the atheromatous plaques. These cytoskeletal changes provide new, possibly useful, biochemical markers for the characterization of smooth muscle cells during early and advanced phases of atheroma formation.     

Development of the origin of the coronary arteries, a matter of ingrowth or outgrowth?

Summary Inconsistencies still exist with regard to the exact mode of development of proximal coronary arteries and coronary orifices. In this regard 15 quail embryos were investigated using a monoclonal anti-endothelium antibody, enabling a detailed study of the development of endothelium-lined vasculature. Coronary orifices emerged at 7–9 days of incubation (Zacchei stages 24–26) and were invariably present at 10 days of incubation (Zacchei stage 27).We never observed more than 2 coronary orifices; these were always single in either of the facing sinuses of the aorta. A coronary orifice was always observed being connected to an already developed proximal coronary artery, which belonged to a peritruncal ring of coronary arterial vasculature. We did not find any coronary orifice without a connection to a proximal coronary artery. Moreover, at 7–9 days of incubation (Zacchei stages 24–26) we observed coronary arteries from the peritruncal ring penetrating the aortic media. In 2 specimen this coronary artery, with a lumen, was in contact with the still intact endothelial lining of the aorta.We conclude that coronary arteries do not grow out of the aorta, but grow into the aorta from the peritruncal ring of coronary arterial vasculature. This throws new light on normal and abnormal development of proximal coronary arteries and coronary orifices.     

CD45 isoform expression during T cell development in the thymus.

Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RA-RO-. The earliest CD3-CD4-CD8-CD19- thymocytes were CD45RO- with 20%-30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RA-RO+. Even the CD4+CD8+ blasts were greater than 90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8- or CD4-CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In contrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RA-RO- or RA+RO- (double-negative thymocytes)----RA-RO+ (double-positive and most single-positive thymocytes)----RA+RO- (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.