Extracellular matrix microfibrils are composed of core proteins coated with fibronectin.

Extracellular proteins of cultured calf aortic smooth muscle cells consist predominantly of microfibrils 10-20 nm in diameter typical of "elastin-associated" microfibrils described in many tissues. Chemical and immunochemical evidence is presented that microfibrils consist of at least two proteins: core protein and fibronectin. Insoluble proteins of the microfibrils were obtained in the form of a pellet and antibodies raised in rabbits against these components. The antisera reacted with the insoluble microfibrillar proteins and with soluble fibronectin in enzyme-linked immunosorbent assay, and immunostained the extracellular microfibrils in cultured cells. An immunoglobulin (Ig) fraction was prepared and absorbed with fibronectin. The absorbed IgG retained its reactivity with the microfibrillar proteins but was no longer reactive with soluble fibronectin. Immunofluorescence studies were carried out using the absorbed IgG and IgG to soluble fibronectin. Both antibodies showed immunoreactive microfibrils in the extracellular matrix of cells in log phase. However, with increasing time in culture, as the cells reached confluence, the immunofluorescence of microfibrils reacting with the absorbed IgG became less intense, whereas that of microfibrils reacting with IgG to fibronectin increased; in confluent cells, essentially no staining was detected with the absorbed IgG, and a dense network of intensely stained microfibrils was seen with IgG to fibronectin. Treatment of these cultures with urea led to partial dissociation of the fibronectin and increased visualization of the microfibrils with the absorbed IgG; double-label immunofluorescence showed that both proteins occurred on the same microfibrils. The localization of immunoreactive sites to the extracellular microfibrils was confirmed by immunoelectron microscopy. Nearly quantitative cleavage with CNBr failed to dissociate the antigenically active fragments of fibronectin from the CNBr fragments of the core proteins of the microfibrils. It was concluded that microfibrils contain core proteins and fibronectin that are codistributed in insoluble, possibly covalently cross-linked, aggregates. The core proteins are first deposited by the cell and, as a function of time in culture, fibronectin gradually coats their surface.

Peroxisomal defects in neonatal-onset and X-linked adrenoleukodystrophies.

Accumulation of very long chain fatty acids in X-linked and neonatal forms of adrenoleukodystrophy (ALD) appears to be a consequence of deficient peroxisomal oxidation of very long chain fatty acids. Peroxisomes were readily identified in liver biopsies taken from a patient having the X-linked disorder. However, in liver biopsies from a patient having neonatal-onset ALD, hepatocellular peroxisomes were greatly reduced in size and number, and sedimentable catalase was markedly diminished. The presence of increased concentrations of serum pipecolic acid and the bile acid intermediate, trihydroxycoprostanic acid, in the neonatal ALD patient are associated with a generalized diminution of peroxisomal activities that was not observed in the patient with X-linked ALD.

Microfibrils, elastic anchoring components of the extracellular matrix, are associated with fibronectin in the zonule of Zinn and aorta.

Microfibrils are striated tubules that play a role in the formation of elastin fibers by providing a scaffold upon which newly synthesized elastin is deposited. Ultrastructural and staining studies also demonstrate microfibrils that terminate where elastin is sparse or absent in basal laminae, plasma membranes, and the collagenous matrix. The most striking accumulation of microfibrils is found in the zonule of Zinn, the transparent and elastic suspensory ligament of the lens, which contains no elastin. Application of immunocytochemical staining with a peroxidase-antiperoxidase (PAP) procedure demonstrates that fibronectin is associated with the microfibrils of the zonule and aorta. Aggregates of microfibrils are identical to oxytalan ('acid enduring') fibers that have been described in peridontal membranes and other sites subject to mechanical stress and they can be found in sites as disparate as the rabbit zonule, rat hepatic stroma and human cardiac papillary muscle, indicating that microfibrils are a widely distributed connective tissue element with a function that extends beyond elastogenesis; their association with fibronectin and localization suggests that they serve as an elastic anchoring component of the extracellular matrix.

Ultrastructure and staining properties of aortic microfibrils (oxytalan).

Microfibrils are the insoluble, 10- to 12-nm components of the extracellular matrix that are involved in elastogenesis. Reports of their ultrastructure vary: they have been described as tubular and beaded and as nontubular filaments that are devoid of any periodicity. Ultrastructurally, microfibrils resemble oxytalan fibers that have been observed in peridontal membranes, skin, and other locations. Whether microfibrils have the staining characteristics of oxytalan is difficult to determine in tissues because available light microscopic stains also stain elastin. Calf aortic smooth muscle cells grown in media without added ascorbate provide a unique model for examining the ultrastructure and staining characteristics of chemically defined microfibrils. Microfibrils are the predominant insoluble extracellular protein in such cultures, which do not deposit collagen or elastin. These studies demonstrate that microfibrils are tubular structures with 10- and 12-nm striations and have the same staining characteristics as oxytalan, reacting with aldehyde fuchsin and orcein after oxidation. Microfibrillar protein is enriched in glutamic and aspartic acids and the electron density of microfibrils is enhanced by fixation in the presence of cationic dyes. In such preparation, microfibrils are made visible within the core of amorphous elastin as well as in regions that are free of elastin. The widespread distribution of microfibrils (oxytalan) indicates that their function extends beyond elastogenesis. Their localization within tissues suggests that they serve as an elastic attachment protein in sites that are subject to mechanical stress.

The infrastructure of aortic elastic fibers.

Elastic fibers are composed of a central core of elastin that is amorphous and electron-lucent in conventional transmission electron micrographs and peripheral microfibrils. A complex infrastructure within the amorphous elastin of mature rat aorta is made visible by fixation and staining with a glutaraldehyde-ruthenium red mixture in phosphate buffer or osmium-ruthenium red in cacodylate buffer. The infrastructure is composed of at least two interlacing but distinct elastic structural components; a framework of circumferentially orientated microfibrils and a three-dimensional meshwork of filaments that permeate the fiber. The latter resembles a reticulum that has previously been observed in freeze-fractured and negatively stained elastin and attributed to the supramolecular organization of elastin. Microfibrils also extend from the core of the elastic fiber into the surrounding matrix where they appear to function as anchoring fibers. These observations indicate that the elastic properties of the arterial wall are an integrated function of both elastin and microfibrils.

Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding.

Culture conditions can modify the composition of the extracellular matrix of cultured calf aortas smooth muscle cells. In the absence of ascorbate the major components of the matrix are microfibrillar proteins; deposition of collagen occurs upon ascorbate supplementation and, with increased time of exposure of cells to ascorbate, collagen becomes the dominant protein of the extracellular matrix (greater than 80%). Collagen accumulation follows a sigmoidal time-course, suggesting that it is a cooperative phenomenon. Covalent crosslinks are not required for collagen accumulation in the matrix. Microfibrillar proteins and increased amounts of proteoglycans and fibronectin accumulate concurrently with collagen but elastin deposition was not observed either with or without ascorbate feeding. Addition of ascorbate leads to a general stimulation of incorporation of [14C]proline into cellular protein and to changes in cell growth parameters and morphology: cell-doubling time decreases from 62 to 47 h and plating efficiency increases approximately fourfold. We conclude that the composition of the extracellular matrix assembled by cultured cells is subject to experimental manipulation and that changes in endogenously deposited matrix may have significant effects on cellular functions.

Glycosaminoglycans in the rat aorta. Ultrastructural localization with toluidine blue O and osmium--ferrocyanide procedure.

Glycosaminoglycans (GAGs) have been implicated in the pathogenesis of sclerotic vascular disease. The localization of GAGs in the rat aorta was examined by two different ultrastructural cytochemical approaches. These procedures are believed to demonstrate 1) anionic sites, with fixatives that contain either toluidine blue or ruthenium red, both cationic dyes, and 2) polysaccharides, proteoglycans, and glycoproteins, with an osmium--ferrocyanide mixture that binds to vicinal diols. Both procedures stain a network of insoluble, 2--8-nm filaments that bridge collagen fibers, elastin, basement membranes, and plasma membranes. These structures resist digestion with chondroitinase ABC and appear to be identical to the filaments that have previously been demonstrated with ruthenium red. Focal 6--12-nm densities are present where filaments intersect. However, the large granules that are made visible with ruthenium red are not seen in toluidine blue or osmium--ferrocyanide preparations. A soluble and relatively amorphous component surrounds the tightly packed bundles of collagen in the media and is preserved and stained by toluidine blue and osmium--ferrocyanide mixtures.

Primary fixation in osmium-potassium ferrocyanide: the staining of glycogen, glycoproteins, elastin, an intranuclear reticular structure, and intercisternal trabeculae.

Primary fixation in an osmium-potassium ferrocyanide (K4Fe(CN)6) mixture combines selective fixation, staining, and extraction of various cellular components; membranes, glycogen, glycoproteins, and elastin are preserved and stained. An intranuclear reticular structure that is composed of 3-6 nm fibers and permeates the entire nucleus, except for the nuclear pores, is demonstrated by electron microscopic examination of tissues prepared in an osmium-potassium ferrocyanide fixative. Condensations of the reticulum parallel the distribution of heterochromatin in interphase nuclei. This preparative procedure also reveals a network of trabeculae that are associated with the cisternae of rough endoplasmic reticulum and connect the parallel cisternae in hepatocytes, plasmacytes, neurons, and pancreatic ancinar cells. The intercisternal trabeculae are associated with both free and bound ribosomes.

Hydrolase activities in the rat aorta. III. Effects of regular swimming activity and its cessation.

It is possible that one of the consequences of regular physical activity could be a change of vascular metabolism. We studied the effects of regular swimming activity on specific activities of aortic hydrolases of male rats. Enzymes included: neutral alpha-glucosidase and lysosomal beta-galactosidase, N-acetyl-beta-glucosaminidase, cathepsin C, acid alpha-glucosidase, and acid cholesteryl esterase. After 8 or 16 weeks of a 1-hour/day swimming protocol, specific activities of four of the six aortic enzymes studied were increased over control levels, increases ranging from 7 to more than 42%. Acid cholesteryl esterase was one of the enzymes most affected by the exercise, increasing 25-30% above control levels. An 8-week sedentary period, after 8 weeks of a swimming regimen, resulted in return of the activity of acid cholesteryl esterase, but not those of the other hydrolases, to control levels. Decreases in body weight, blood pressure, and serum lipid levels also occurred in the swimming rats. Weight reduction per se was excluded as an explanation for the increases in aortic enzymes or decrease in serum cholesterol found with swimming. These findings show that regular physical activity is yet another factor with discrete and significant effects on the catabolic activity of vascular tissue.

Immunocytochemical localization of hepatic legandin and Z protein utilizing frozen sections for light and electron microscopy.

Ligandin (glutathione-s-transferase) and Z protein are soluble hepatocellular proteins that are involved in the transfer of organic ions, including bilirubin and some hormones and carcinogens from the plasma to the liver. The intracellular distribution of ligandin and Z protein was studied by applying the peroxidase-antiperoxidase procedure of L. A. Sternberger (Immunocytochemistry, Prentice Hall Inc., 1974) to paraffin sections and free-floating 10-micrometers frozen sections that were processed for both light and electron microscopy. Ligandin and Z protein were localized to the cytosol of hepatocytes in association with smooth endoplasmic reticulum (SER), but no reaction product was present between cisternae of rough endoplasmic reticulum. Penetration of reagents was enhanced in 10-micrometers frozen sections and the preservation of subcellular structures was equivalent to thicker, unfrozen sections.

Hydrolase activities in the rat aorta. I. Effects of diabetes mellitus and insulin treatment.

Vascular disease in diabetics could arise in part from altered vessel wall catebolism. Specific activities of hydrolases in aortic smooth muscle cells from rats with streptozotocin-induced diabetes were measured. Enyzmes included: neutral alpha-glucosidase, alpha-mannosidase, and lysosomal N-acetyl beta-glucosaminidase, beta-galactosidase, cathepsin C, acid alpha-glucosidase, and acid cholesteryl esterase. After 4,8, and 11 weeks of diabetes, activities of all enzymes studied were decreased significantly in diabetic vessels, decreases ranging from 15% for cathepsin C to 62% for alpha-mannosidase. After 3 weeks of diabetes, insulin treatment for 1 week restored enzyme levels to normal. After 7 weeks of diabetes, 1 week of insulin treatment did not restore enzyme levels fully to normal (acid cholesteryl esterase was unchanged); 4 weeks of insulin did. Acid phosphatase and N-acetyl beta-glucosaminidase activities were reduced markedly in histochemical studies of diabetic aortas at all time periods and were restored by insulin treatment. Alloxan-induced diabetes gave results similar to those with streptozotocin. Significant decreases of aortic hydrolase activities, including those of lysosomes, occur in experimental diabetes mellitus and could contribute to accumulation of substrates in vascular smooth muscle cells.

Hydrolase activities in the rat aorta. II. Effects of hypertension alone and in combination with diabetes mellitus.

Hypertension is an important risk factor for atherosclerosis and often occurs in association with diabetes mellitus. Specific activities of hydrolases in homogenates of aortas from rats with renal-clip hypertension, normotension following a period of hypertension, and hypertension combined with streptozotocin-induced diabetes mellitus were measured. Enzymes included: neutral alpha-glucosidase, and lysosomal N-acetyl-beta-glucosaminidase, beta-galactosidase, cathepsin C, acid alpha-glucosidase, and acid cholesteryl esterase. After 6 or 12 weeks of hypertension, specific activities of all enzymes measured were significantly increased, levels ranging from 24% above normal for cathepsin C to 351% above normal for N-acetyl-beta-glucosaminidase. Six weeks of normotension following 6 weeks of hypertension resulted in restoration to normal of four of the six enzyme activities; the remaining two enzymes were significantly below normal levels. Combined hypertension and diabetes mellitus showed smooth muscle cell levels of four of the five hydrolases measured to be significantly lower than those present with hypertension alone. In every instance, histochemical studies of aortas showed acid phosphatase and N-acetyl-beta-glucosaminidase activities which corresponded to the biochemical findings. These findings indicate profound and discrete effects of two clinical risk factors on vascular smooth muscle cell lysosomes.

Lipid accumulation in human aortic smooth muscle cell lysosomes.

Lipid deposition is a central feature of the human atherosclerotic lesion. Deficient lysosomal lipolytic activity has been implicated as a pathogenetic factor in atheroma formation. Cytochemical and ultrastructural examination of the abdominal aortas of 2 normal young males, ages 11 and 23, demonstrates lipid accumulation with lysosomes of intact mural smooth muscle cells. This appears to be an early stage in the process which eventually results in an overloading of lysosomes and the formation of lipid-laden foam cells.

Lysosomes and the sclerotic arterial lesion in Hurler's disease.

A case of Hurler's disease in a mentally retarded, six year old boy is reported. In Hurler's disease a lysosomal hydrolase, l-iduronidase, is deficient, and consequently undegradable mucopolysaccharide accumulates within lysosomes in many tissues. Severe occlusive coronary artery disease and sclerotic aortic lesions are common in very young patients, although their serum lipid and blood pressure levels are normal. Vascular collagen and elastin is increased, but little or no stainable lipid is present. Electron microscopy shows that aortic smooth muscle cells are distended by vacuoles, appearing empty in formalin fixed tissues, that identify them as the "gargoyle" cells in the proliferative lesion. The presence of a basic lysosomal defect and the absence of other contributing metabolic factors suggest that accumulation of an excess of undegradable substrate within smooth muscle lysosomes may be an initiating event in the development of proliferative sclerotic vascular lesions.

Arterial lysosomes and connective tissue in primate atherosclerosis and hypertension.

The cellular events that occur in the vessel wall consequent to changes in endothelial permeability result in the progression of vascular disease, particularly atherosclerosis. Female rhesus monkeys were fed an atherogenic diet or were made hypertensive for 6-8 months; and their vessels were then compared with vessels from control monkeys. Length-defined segments of coronary vessels, the thoracic aorta, and the abdominal aorta showed significant increases in total connective tissue in the atherosclerotic and hypertensive groups; pulmonary vessels did not. The diseased aortic segments had increased levels of two lysosomal enzymes, acid phosphatase and beta-N-acetylglucosaminidase; pulmonary vessels were not diseased and did not show these changes. Coronary vessels from the atherosclerotic and hypertensive groups did not show an increase in enzyme levels on biochemical measurements, but focal accumulations of lysosomes were identified by cytochemical techniques. In atherosclerotic lesions, a doubling of cholesterol and more than a tenfold increase in cholesterol ester were found. These connective tissue and lysosomal changes are early features of primate vascular disease and may result from the accumulation of excessive substrate (cholesterol ester) in the lysosomes of vascular smooth muscle cells.