Amyloid A gene family expression in different mouse tissues.

Serum amyloid A (SAA) is a major acute-phase reactant and apoprotein of high density lipoprotein (HDL). SAA is encoded by a family of three active genes. We examined hepatic expression and searched for extrahepatic expression of the three SAA mRNAs after injection with casein or LPS. Studies using an SAA cDNA, which detects all three SAA mRNAs, revealed that after casein injection liver SAA mRNA was elevated approximately 1,000-fold. Adrenal gland expressed SAA mRNA at a low level (0.5% of hepatic level), and was the only extrahepatic tissue with elevated SAA mRNA after casein injection. The small intestine, primarily the ileum, and the large intestine of unstimulated control animals contained 5- and 15-fold higher SAA mRNA levels than control liver. LPS also elevated liver SAA mRNA approximately 1,000-fold. However, in contrast to casein injection, every extrahepatic tissue examined expressed SAA mRNA. Lung and kidney contained 2-5% and large intestine contained nearly 10% of SAA mRNA levels found in liver RNA. SAA mRNA levels were lower in the remaining tissues and ranged from 0.1% in the brain and pancreas to 1.0% in the small intestine, with the ileum containing 50-fold more than the duodenum. Analysis of liver with SAA1, SAA2, and SAA3 mRNA-specific oligonucleotide probes revealed that SAA1 and SAA2 mRNA were elevated approximately 50-fold higher than SAA3 mRNA after casein administration. LPS, however, induced all three SAA mRNAs equally. In extrahepatic tissues, SAA1, SAA2, and SAA3 mRNAs were expressed differentially and can be grouped into three general classes: tissues expressing all three genes, tissues expressing SAA1 and SAA3, and tissues expressing predominantly or only SAA3.

Expression of the third member of the serum amyloid A gene family in mouse adipocytes.

Three homologous genes that code for three related proteins comprise the serum amyloid A (SAA) family in the mouse. Endotoxin induces equally vigorous expression of mRNAs for the three SAA genes in liver. In extrahepatic tissues SAA1 and/or SAA2 mRNAs have been found only in kidney and intestine, however, SAA3 is expressed in all extrahepatic tissues thus far examined. This observation raised the question: is SAA3 mRNA expressed by a single cell system dispersed throughout all tissues, or by differentiated cells of each tissue? This question was explored in various tissues by in situ hybridization with a single-stranded cRNA probe specific for SAA3 mRNA. We found expression in the liver of SAA3 mRNA by other cells as well as by hepatocytes. A common feature among extrahepatic tissues was SAA3 mRNA expression in adipocytes. SAA3 mRNA was also found in two nonadipose cells, Leydig cells of the testis, and some of the cells located in parafollicular zones of the spleen.

Amyloidogenesis. One serum amyloid A isotype is selectively removed from the circulation.

The deposits of fibrils found in amyloidosis of the A type are derived from only one of the three serum amyloid A (SAA) gene products, namely SAA2. In order to explore the mechanism of SAA isotype-specific amyloid protein AA deposition, the molecular kinetics of the serum amyloid proteins were examined in CBA mice during casein induction of amyloidosis. The presence of SAA mRNA in spleen was searched for; hepatic SAA1 and SAA2 mRNA levels, rates of specific protein synthesis and secretion by hepatocytes, and serum levels were measured during a 20-d period of amyloid induction. We observed the following: small amounts of amyloid substance appeared in the spleen by day 5 and increased steadily over the ensuing 15 d to occupy nearly 30% of splenic volume by day 20. No SAA mRNA was detected in spleen at any time during induction of amyloid formation. Total serum SAA levels peaked 1 d after we began casein treatment, and thereafter declined. This decline was accounted for entirely by a dramatic fall in SAA2, while SAA1 levels remained nearly constant throughout. The ratios of hepatic SAA2:SAA1 mRNA, as determined by in vitro translation, remained constant during the 20-d period, as did amounts of SAA1 and SAA2 synthesized and secreted by freshly isolated hepatocytes. These data indicate that the deposition of amyloid A protein derived from SAA2 is not due to local SAA production in spleen, nor excessive SAA2 production compared with SAA1, but involves the selective and accelerated removal of SAA2 from the circulating pool of both SAA1 and SAA2.

Murine tissue amyloid protein AA. NH2-terminal sequence identity with only one of two serum amyloid protein (ApoSAA) gene products.

Amyloid protein AA is the presumptive fragment of an acute phase serum apolipoprotein, apoSAA. Two major murine apoSAA isotypes (apoSAA1 and apoSAA2) have been identified. The NH2-terminal amino acid sequences of purified murine apoSAA1 and apoSAA2 have been examined and compared with that of murine amyloid protein AA. Our results indicate that apoSAA1 and apoSAA2 are separate gene products and that amyloid protein AA has NH2-terminal amino acid sequence identity with only one of these isotypes, namely apoSAA2.

Basal lamina: the scaffold for orderly cell replacement. Observations on regeneration of injured skeletal muscle fibers and capillaries.

To explore in detail the relationships between basal lamina (BL) and regenerating cells, we have studied the reconstruction of skeletal muscle fibers and their associated capillaries in portions of rat and rabbit skeletal muscles after injury with either freezing, ischemia, or in situ autografting. Each type of injury produces complete necrosis of cells. The BL, however, remains intact in the area of injury and maintains a "map" of the outline of the spatial relationships between muscle fibers and capillaries. Repopulation of the defect with new cells occurs primarily along the old BL. The spatial relationship between cells, as it existed before injury, is thus reestablished. This process appears to be aided by the ability of each category of regenerating cells to grow along the cell-supporting surface of its own BL. The regenerating cells of muscle fibers and capillaries frequently form a new layer of BL. It is of the usual thickness and is deposited primarily along the outer surfaces of plasma membranes in locations in which the new cells are separated from the old BL. Where an old layer of BL is present overlying a newly formed layer, the old layer may be retained or it may be removed. Removal of redundant BL is probably mediated by interstitial cells which embrace the outside surfaces of BL of regenerated skeletal muscle fibers and capillaries.

Collagen has a discrete family of reactive hydroxylysyl and lysyl side-chain amino groups.

Several cross-linking substances which derive from lysine have been isolated from hydrolyzates of collagen and elastin. This observation suggests that certain lysyl side chains exhibiting enhanced reactivity and unique configuration may participate in cross-linking. In native collagen there is a small family of lysyl and hydroxylysyl side chains which exhibit a high propensity of Schiff base formation.

Plasma clearance kinetics of the amyloid-related high density lipoprotein apoprotein, serum amyloid protein (apoSAA), in the mouse. Evidence for rapid apoSAA clearance.

The plasma clearance kinetics of the amyloid-related high density lipoprotein (HDL) apoprotein serum amyloid protein (apoSAA) was examined in BALB/c mice by two different methods, using labeled 125I-apoSAA-rich HDL and unlabeled plasma apoSAA (clearance monitored by radioimmunoassay). The plasma half-life of apoSAA, estimated by both methods, was on the order of 75-80 min, as compared with a value of approximately 11 h for mouse apoA-I. In trace-labeling studies, the rapid plasma clearance of both major 125I-labeled apoSAA isotypes was observed; this metabolic behavior was unique to these polypeptides among HDL apoproteins. The property of rapid plasma clearance was lost upon purification and reconstitution of 125I-apoSAA with HDL, indicating that this property is labile to denaturing conditions. Studies aimed at determining the metabolic fate of 125I-apoSAA gave no evidence for either the selective excretion of 125I-apoSAA or clearance to unique tissue sites as compared with other 125I-HDL apoproteins.

What role(s) may extracellular matrix, particularly heparan sulfate, play in amyloid of Alzheimer's disease.

The authors of this article present and attempt to substantiate the thesis that proteoglycan(s), mainly heparan sulfate, are an important ingredient in the pathogenesis of the amyloid found in persons with Alzheimer's disease. Evidence presented indicates that glycosaminoglycans are regular constituents of many amyloid substances including that of senile plaques and congophilic angiopathy of Alzheimer's disease. It is proposed that the proteoglycans play a role in amyloidogenesis by one or a combination of the following mechanisms: 1) inducing amyloid fibrils containing a predominant beta-pleated sheet structure, 2) influencing amyloid deposition to occur at specific tissue sites and/or 3) prevent amyloid degradation.

Primary structure of duck amyloid protein A. The form deposited in tissues may be identical to its serum precursor.

The amino acid sequence has been determined for the major protein that accumulates in amyloid fibrils in tissues of the Pekin duck. With the exception of 16 residues at the amino terminus, this 106-residue protein is homologous with human serum amyloid protein A (104-residue apoSAA), which is the putative precursor of the 76-residue protein that accumulates in human patients with amyloidosis. Duck serum is shown to contain a protein that is immunologically related and approximately equal in size (12 kDa) to the deposited form in ducks. These results indicate that proteolytic processing of the precursor is not a necessary step in the deposition of amyloid fibrils, at least in the duck.

Replication in restenotic atherectomy tissue.

Previously, we demonstrated that replication in restenotic coronary atherectomy specimens was an infrequent and modest event. In general, this data was interpreted with caution, as immunocytochemistry for the proliferating cell nuclear antigen (PCNA) was used to subjectively assess proliferation and most of the tissue specimens were resected more than 3 months after the initial interventional procedure. The purpose of the present study was to use a more sensitive method of detecting replication, in situ hybridization for histone 3 (H3) mRNA, to determine the replication profile of human directional atherectomy specimens. Restenotic directional coronary atherectomy specimens from lesions that had undergone an interventional procedure within the preceding 3 months were studied. In addition, larger atherectomy specimens from peripheral arterial lesions were assessed to ensure that pockets of replication were not being overlooked in the smaller coronary specimens. We found evidence for replication in tissue resected from 2/17 coronary and 9/12 peripheral artery restenotic lesions. In contrast, 3/11 specimens resected from primary lesions of peripheral arteries also expressed H3 mRNA. We estimated that the maximum percentage of cells that were replicating in restenotic coronary, restenotic peripheral and primary peripheral artery tissue slides to be <0.5, < or =1.2 and <0.01%, respectively. Replication was found in tissue specimens resected both early and late after a previous interventional procedure. For specimens with >15 replicating cells per slide we found high levels of focal replication. Therefore, cell replication, as assessed by the expression of H3 mRNA, was infrequent in restenotic coronary artery specimens, whereas peripheral restenotic lesions had more frequent and higher levels of replication regardless of the interval from the previous interventional procedure. For all specimens the percentage of cells that were replicating was low, however focal areas with relatively high replication indices were presented. Although replication was more abundant in restenotic lesions it does not appear to be a dominant event in the pathophysiology of restenosis.

Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation.

Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.

Type III hyperlipoproteinemia and atherosclerosis: a case report and re-evaluation.

a patient with type III hyperlipoproteinemia (HLP) who had been followed for several years at the Northwest Lipid Research Clinic died suddenly. Autopsy findings included severe atherosclerosis of the native coronaries as well as atherosclerotic involvement of saphenous vein bypass grafts. Nevertheless, there was a striking absence of atherosclerosis of the aorta or of any other noncoronary artery. These results are compared with those of previously reported cases of type III HLP, and the findings are analyzed in terms of the current concepts of the pathogenesis of type III HLP. It is suggested that there remain factors yet to be uncovered, which determine the degree and location of atherosclerotic disease involvement of the vascular tree.

SAA, an apoprotein of HDL: its structure and function.

The putative precursor of amyloid protein AA appears in serum as an apoprotein (apoSAA) of heavier HDL fractions of lipoproteins found in humans and mice. ApoSAA is found by precipitation with specific anti-AA antibodies to be on a particle that also carries apoA-I and some C-apoproteins. In endotoxin-treated mice a lipoprotein fraction with about 2 moles of apoSAA to 1 mole of apoA-I can be isolated, an apoSAA-carrying subset of HDL being thus suggested. In mice, rapid clearance from plasma of native apoSAA compared to apoA-I suggests a special function for apoSAA. The C-terminal amino acid portion of apoSAA may play a role in this function.

Trauma, high density lipoproteins, and serum amyloid protein A.

Several apoproteins not readily detectable in normal serum lipoproteins were found in markedly increased amounts in the lipoprotein density interval 1.125-1.21 g/ml (HDL3) of serum obtained from victims of severe trauma. Following electrophoretic separations, they were shown to be isotypes of the acute-phase protein serum amyloid A (SAA) by immunoreaction with antiserum to the structurally related tissue amyloid protein A (AA). The two principal apoSAA isotypes in the HDL3 of trauma patients appear to be identical to the two principal isotypes (apoSAA1 and apoSAA2) previously isolated from the HDL3 of pooled serum representing an unselected patient population. Concentrations of apolipoproteins A-I and A-II in the HDL3 of the trauma patients were significantly lower than in the HDL3 of normal serum. Evidence is presented that several recently described 'new' families of HDL apoproteins, all of an acute-phase nature, are SAA apoproteins, which are emerging as sensitive indicators of tissue damage.

Origins of human atherosclerotic plaques. The role of altered gene expression.

The dramatic rise and equally dramatic fall in the mortality from coronary heart disease in the 20th century is only partly explained. This article reviews the development of our ideas concerning possible pathways other than lipids that might play a role in the development of human atherosclerosis alone or in combination with one or more of the usually considered risk factors. In some instances, such as that of cigarette smoking, the proposed concept regarding genetic alterations in vascular smooth muscle suggests a mechanism for development of at least some of the lesions. Recent studies have shown that an aberration in platelet-derived growth factor gene expression is unlikely to be a factor in proliferation of smooth-muscle cells. Aberrant expression of other oncogenes or some as yet unknown virus remain as possible explanations of some of the incidence of atherosclerosis and its consequent coronary heart disease.